The impact of maternal microbial influences on the early choreography of the neonatal calf microbiome were investigated. Luminal content and mucosal scraping samples were collected from ten locations in the calf gastrointestinal tract (GIT) over the first 21 days of life, along with postpartum maternal colostrum, udder skin, and vaginal scrapings. Microbiota were found to vary by anatomical location, between the lumen and mucosa at each GIT location, and differentially enriched for maternal vaginal, skin, and colostral microbiota. Most calf sample sites exhibited a gradual increase in α-diversity over the 21 days beginning the first few days after birth. The relative abundance of Firmicutes was greater in the proximal GIT, while Bacteroidetes were greater in the distal GIT. Proteobacteria exhibited greater relative abundances in mucosal scrapings relative to luminal content. Forty-six percent of calf luminal microbes and 41% of mucosal microbes were observed in at-least one maternal source, with the majority being shared with microbes on the skin of the udder. The vaginal microbiota were found to harbor and uniquely share many common and well-described fibrolytic rumen bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing rumen and reticulum with microbes important to the nutrition of the adult animal. Colonization of the ruminant gastrointestinal tract (GIT) begins during birth and progresses through until a climactic community establishes8. Once this climax community is reached, the GIT microbiome is generally considered to be stable, excepting for changes in diet, physiological state, or health status13. Adult microbial diversity profiles do, however, differ along the GIT exhibiting biospatial circumscriptions that separate anatomical locations16. Similarly, there are also differences known to exist between microbes occupying the lumen, and those found associated with the mucosal-epithelia of the GIT17. These differences are likely due to the varying niches imparted by localized variations in nutrient profiles, pH, transit rates, host physiology, immune cell populations, and the differing interactions between host-epithelia and symbiotic bacteria20. For example, pH varies through the rumen GIT being lowest in the abomasum (~pH 2–4) and highest in the distal GIT (~pH 6)21; The rate at which digesta transits through the GIT is affected by diet, but is generally faster in the small intestine than the abomasum22 and slowest in the rumen and large intestine (cecum & colon)23; while a steep oxygen gradient runs through the epithelium to the hypoxic lumen24. Each of these factors refine the colonization potential at each site limiting occupation to the microbes for which these collection of conditions fall within their fundamental niche leading to described and hypothesized impacts on the microbial composition27. How each location of the ruminant GIT is seeded and the ecological dynamics underpinning their succession remains to be described. Findings from studies on human neonatal colonization28 have led those studying cattle to hypothesize that the earliest exposure of neonatal calves to microorganisms also occurs during parturition, when the calf is exposed to microbes occupying the dam’s vagina29. The calf may subsequently be exposed to microbes residing on the dam’s skin28, and recent evidence of a colostrum microbiota have led to the hypothesis that it may also provide an early source of microbes to the neonatal GIT32. Consistently, the DNA of common GIT microbes have been reported at low relative abundances in breast milk of cattle, goats, and humans37. Important microbial groups and critical nutritive functions such as hemicellulolytic (3 d), pectinolytic (3 d), cellulolytic (2–3 w), and methanogenic (1–3d) activities have been shown to emerge early in the rumen38. However, the successional development of the rumen appears to be far more protracted with substantial dynamism initially before settling on a climactic adult-like state8, similar to what has been described in humans39. For ruminants, these changes may partially reflect dietary transitions from colostrum to milk or milk replacer, and then to a progression of solid diets given in production settings to adapt the animal to an adult feeding regimen17. Successional changes in the GIT biochemistry brought about by these dietary transitions, as well as each colonizing species’ metabolic activities are also likely to impact community membership41. It is also likely that stochastic processes consistent with the unified neutral theory of biodiversity42 are involved, though the degree and duration are unclear.The microbiota of the GIT are of critical importance to the nutritive processes, health, and development of all known mammals, including cattle and other ruminantsBecause the actual influence of maternal sources of microbes to the early succession of the calf GIT has neither been experimentally determined nor quantified, we set out to determine the relationships among the microbiota of the dam’s vagina, skin, and colostrum to the early successional development of the calf microbiome. The dam’s udder was used as our representative skin microbiota because under typical conditions it is more likely to directly interact with the calf’s GIT than other skin surfaces. Additionally, because colostrum could potentially contain autocthonous microbiota from the mammary alveolus or intralobular duct, along with auto- or allocthonous microbiota picked up from the teat or surrounding skin , we wished to compare the outer skin of the udder to colostrum.All experimental procedures were performed at Fair Oaks Farms  and University of Illinois Veterinary Medicine Research Farm  under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois (protocol #13096). Twelve healthy male Holstein calves were separated from their dam within 15 minutes of birth, dried off with a hair dryer and had their navel disinfected with iodine solution. No vaccination was given.nd lactation cow, 2) single calf, 3) colostrum quality assessed by a refractometer (Misco® DD-1) >20° Brix, corresponding at a minimum of 50 mg/L of IgG, 4) colostrum produced was at least 4 L. After birth, each calf received 2 liters of colostrum aseptically collected from the respective dam within 1 hour. After first feeding, colostrum was kept refrigerated and warmed up before administration. Calves were offered first milking colostrum again on the second feeding at 4 hours after birth. Calves were subsequently fed three times a day with a non-medicated milk replacer (Agrimaster®) for the remainder of the experiment and had access to fresh water ad libitum.Calves were recruited to the study if their dam fulfilled the following criteria: 1) 2All calves were monitored, and fed separately in individual 10′ × 10′ indoor pens of the same barn. Pens were bedded with clean sawdust and straw. Only 2–4 calves were present in the barn at any one time and pens were separated from one another by ≥10 ft. Dams were not cohoused, nor had ever previously been housed in the same barn as calves. Milk replacer intake and health checks were recorded daily. Health checks included body temperature, animal behavior, respiration, and fecal score . Body weight was monitored every other day in order to adjust milk replacer intake accordingly (feeding rate: 10% of body weight). Extruded fecal samples were carefully collected from calves to minimize potential contamination at days 0, 1, 2, 3, 4, 5, 7, 14, and 21 after colostrum administration.Three calves were euthanized at each of age 1, 3, 7 and 21 days for necropsy, and luminal digesta and mucosal scraping samples were collected from the pharynx, rumen, reticulum, omasum, abomasum, duodenum, jejunum , ileum, and colon. Samples were also collected from the dam’s colostrum, outer udder skin, and vaginal scrapings immediately at calving. After collection, all samples were immediately frozen in liquid nitrogen and kept at −80 °C until analysis.Samples were processed for DNA using the PowerSoil 96-well DNA Isolation Kit  strictly adhering to the manufacturers instructions. The V3-V4 region of the 16S rRNA gene was PCR-amplified using the KAPA HotStart PCR Kit : 10 µL Kappa HotStart Mastermix, 6 µL molecular-grade water, 1 µL of each forward and reverse primer, and 2 µL sample DNA. The Hot Start protocol was as follows: 95 °C for 3 min; 5 cycles of denaturation at 98 °C for 20 sec, annealing at 52 °C for 30 sec, elongation at 72 °C for 45 sec; 25 cycles of denaturation at 98 °C for 20 sec, annealing at 60 °C for 30 sec, elongation at 72 °C for 45 sec; followed by storage at 4 °C. Primers amplified the V3-V4 region of the microbial 16S rRNA gene, and included the MiSeq adaptors , the sample index/barcodes, the two-nucleotide linker, and the primers 341 F 5′-CCTACGGGAGGCAGCAG-3′and 806 R 5′-GGACTACHVGGGTWTCTAAT-3′.PCR amplicon concentration and quality was checked on a TapeStation Bioanalyzer  using the D1000 Screentapes with 3 µl of D1000 buffer and 1 μl of PCR product in each tube. Samples were then diluted with molecular water and pooled at equimolar concentrations of amplicon. The pooled amplicons were gel purified from a 1.5% agarose gel using the QIAquick gel extraction kit  according to kit instructions. Sequencing was performed on an Illumina MiSeq  using a 2 × 251 nt sequencing approach with a 500 cycle v2 kit. A no-template (molecular grade water) negative control and a custom 32 member mock community positive control were also processed and sequenced. Resulting FASTQ files, and can be found in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA314799.43 with default assembly parameters. Assembled sequences were then curated using in-house scripts and mothur v.1.3544 as follows: First, sequences were trimmed after any homopolymer >8 nt long using an in-house script. This was justified as homopolymer length is not directly tied to whole sequence error rate in Illumina MiSeq data45. Mothur was then used to remove sequences which were <400 nt or >580 nt, or which contained any ambiguous bases (N). Sequences were aligned using mothur to the Silva v119 reference database, which contains bacterial and archaeal 16S rRNA gene sequences using the Wang algorithm46. Chimeras were identified and removed using the mother-integrated version of UCHIME47, and sequences were pre-clustered by 2 base differences as previously described48. Sequences were classified using mother-implemented RDP classifier using the Silva v119 taxonomy database at 80% confidence cutoff46. Taxonomy was used to bin sequences into Operational Taxonomic Units (OTUs) at the genus-level, which is the lowest limit of taxonomic resolution available from the Silva taxonomy. Three OTUs detected as contaminant in blank and mock controls  were removed prior to analyses.Samples were demultiplexed using CASAVA software v1.8.2 . Paired-end 16S rRNA sequences for each sample were assembled into contiguous sequences (contigs), using PANDAseq.53 values are presented as group mean. Linear Discriminant Analysis was run to detect discriminatory OTUs, using Wilcoxon Rank Test to determine significance. The R statistical package ggplot2 was used to create heatmaps54. PRIMER ver. 655 was used to determine Bray-Curtis Dissimilarities among samples, create a Non-metric Multi-Dimensional Scaling (nMDS), perform ANOSIM and PERMANOVA calculations, and determine Ewans-Caswell statistics for assessing the deviations from the assumptions of neutrality42. One way ANOVA was used to test for significant differences among taxa, in the Bray-Curtis similarities (1 – dissimilarity) between maternal and calf GIT samples, and between sample types for Ewans-Caswell V statistics. All statistics were corrected for multiple tests using the Benjamini-Hochberg FDR approach where necessary.Sequences were subsampled to the lowest number (min. 2500) of reads for each statistical analysis. The diversity measures ACE, CHAO, Inverse Simpson, Good’s Coverage, Shannon-Weiner diversity, and UniFracMethanobrevibacter.A total of 2,511,755 high-quality sequences were clustered to give a total of 1,213 OTUs. Among OTUs, 75% of taxonomic classifications were supported at the genus level and Good’s estimates of coverage indicated we had captured sequences from the majority of OTUs present . Firmicutes were also found to have greater relative abundances in the colon (avg. relative abundance = 55%) and feces (avg. 41%) than other GIT locations (P < 0.05). Bacteroidetes conversely had greater relative abundances in the reticulum (avg. 26%), rumen (avg. 26%), omasum (avg. 21%), and abomasum (avg. 25%) than in either the colon (avg. 13%) or feces (avg. 13%) (P < 0.05). Throughout the GIT, Bacteroidetes were predominantly of the genera Bacteroides (avg. 9%) and Prevotella (avg. 2%) although the relative abundances of these genera varied with GIT location and between luminal and mucosal microbiota averaging 18% and 20% in mucosal samples of the reticulum and proximal jejunum, respectively , and reticulum (avg. 12% in lumen), and in the luminal content of the omasum (avg. 10%), and abomasum (avg. 15%). Escherichia spp. outnumbered Bacteroides spp. in the omasum (avg. 14%) and abomasum (avg. 21%) mucosa , being outnumbered by Pseudomonas spp. in the mucosa (avg. 5.5%) , but were outnumbered by Prevotella and the Rikenellaceae RC9 group (avg. 10.1%) in the proximal jejunum mucosa. Streptococcus spp., which had similar relative abundances in the duodenum (avg. 2.7% in lumen and 3.1% in mucosa), had the highest relative abundance in the lumen of the proximal (avg. 3%) and medial jejunum (avg. 1.7%) and colon (avg. 6.9%) , in both the lumen (avg. 2.8%) and mucosa (avg. 9.8%) of the ileum, the mucosa of the colon (avg. 15.2%), as well as in feces (avg. 16.9%)  , Firmicutes avg. 29%), and Bacteroidetes (avg. 23%) were the most prevalent phyla throughout the early calf GIT , and with calf age (pseudo F = 4.25 P = 0.001). Luminal and mucosal samples were only different when nested within each GIT location (pseudo F = 4.21 P = 0.001), although a weighted UniFraq supported overall differences between the two sample types (Table P > 0.2), or between the colon and feces . Other relationships were determined among anatomically connected locations, including the the rumen, reticulum and omasum , and the ileum and each portion of the jejunum . The ileum was most similar to the distal jejunum and progressively less similar to the medial and proximal jejunum. All other GIT regions differed significantly.Multiple measures of β-diversity (including phylodiversity) indicated the strongest differences to be among GIT locations  including the only P < 0.001) sharing just 11 OTUs (<7% of observed). Both colostrum and udder skin samples exhibited even greater dissimilarity when compared to vaginal samples .The mean α-diversity indices for maternal sources of microbiota indicated the udder skin had the greatest richness and diversity, followed by colostrum, and then vaginal samples Table . MeasureLactococcus was found to have the greatest relative abundance (average 13.6%) in colostrum prior to refrigeration, however, its relative abundance decreased to less than 0.5% in all colostrum samples following refrigeration. Similar changes were seen in Staphylococcus spp. (avg. 1.3% to 0.12%). The decreased relative abundances of these genera following refrigeration corresponded to 1.5–2 fold increases in numerous, mostly unclassified genera of the Proteobacterial phylum. The genera Pseudomonas (avg. 2.3%), Bacteroides (avg. 1.4%), Streptococcus (avg. 1.3%), Bifidobacterium (avg. 0.6%), Akkermansia (avg. 0.4%), Escherichia (avg. 0.3%), and Lactobacillus (avg. 0.2%) were also prevalent along with unresolvable members of the bifidobacteriaceae family (avg. 0.1%) but appeared to be unaffected by refrigeration.Colostrum was determined to predominantly harbor bacteria of the phyla Proteobacteria (avg. 42%), Firmicutes (avg. 22%), and Bacteroidetes avg. 21%) , Arthrobacter (avg. 4.9%), Pseudomonas (avg. 4.1%), Streptococcus (avg. 3.3%), Acinetobacter (avg. 2.2%) and Staphylococcus (avg. 2.0%) , the Rikenellaceae RC9 group (avg. 4.9%), Ruminococcus (avg. 1.1%), and Butyrivibrio (avg. 1.1%) , Bacteroidetes , Actinobacteria  and Proteobacteria  , several had greater or equivalent Bray-Curtis (BC) similarity to maternal colostrum or vaginal samples  among all maternal sources at day 1, with the later two GIT regions being initially more similar to colostrum (P < 0.05), but by day 21 all had equivalent BC similarity to the vagina and udder skin and greater BC similarity than colostrum (P < 0.05). With the exception of the proximal and distal jejunum, the ileum, and the colon all mucosal samples did exhibit the greatest BC similarity to maternal udder skin samples by day 21, although the reticulum and omasum had equivalent BC similarity to maternal vaginal samples. Instead the microbiota of the proximal jejunum were more similar to those of the dam’s vagina at days 1, 3 and 21, and the distal jejunum was more similar to colostrum samples at all ages. The ileum was initially more similar to maternal udder skin samples but by day 7 had identical BC similarity to second-fed colostrum and was more similar to first-fed colostrum at day 21 (P < 0.05). The colonic mucosa was initially most similar to the maternal udder skin, but became most similar to second-fed colostrum at days 3 and 7 before becoming more similar to the dam’s vagina at day 21 (P < 0.05) and this pattern was largely reflected in fecal samples.Because OTUs shared between the different calf GIT regions and the maternal sources varied in their abundances, measures of β–diversity indicated that, although all calf GIT and fecal samples were significantly different from each maternal reservoir , the deviation statistic (V) of which indicated greater dominance by select taxa than would be expected in a neutral model. Of maternal sources, colostrum exhibited the largest deviation from the assumptions of neutral theory (P < 0.05), and the udder skin exhibited the least numerically, though was not statistically different from the vagina (P = 0.23). The udder skin was not significantly less neutral than any GIT region, and the vagina was only different from the reticulum and omasum (P < 0.05), although trended toward a difference from the rumen (P = 0.09).Ewans’-Caswell60, skin61, and colostrum62. The microbiota determined in our study are each consistent with those reported previously.Several studies have previously described the microbiota of the cattle vaginaAggregatibacter or Streptobacillus spp., as previously described by Swartz et al.56, however, many of the other genera described in that study, and most described in separate studies of Nellore cattle by Laguardia-Nascimento et al.57 were observed. These included Myroides, Flavobacterium, Lactobacillus, Campylobacter, and Escherichia spp., as described by Swartz and colleagues56 and Alistipes, Bacillus, Ruminococcus, Rikenella, and Prevotella spp. described by Laguardia-Nascimento and colleagues57. Both studies have now reported Bacteroides, and methanogenic archaeal populations in the cattle vagina57. These microbial groups have also been recently detected in the uterus of dairy cattle64. A fourth study investigating the vaginal microbiota associated with bovine necrotic vulvovaginitis58 only reported phylum-level classifications. In that study the authors described: i) Bacteroidetes and Proteobacteria levels consistent with this study and the studies of Swartz et al., and Laguardia-Nascimento and colleagues57; ii) Firmicutes at levels consistent with this study and that of Laguardia-Nascimento and colleagues57; and iii) Fusobacteria levels consistent with that of Swartz and colleagues56.In our study, vaginal microbiota were not found to be dominated by 61 that used both cultivation and culture-independent techniques and reported Corynebacterium spp.  and Staphylococcus spp.  to be abundant along with lower abundances of Arthrobacter (A. gandavensis), Pseudomonas (P. sp.), and Streptococcus (S. equinus) spp.Microbiota of the udder skin, were also consistent with a previous survey of cattle teat skin microbiota62, as well as to a more recent study in goats31. Both31 and32 reported Lactococcus  and Lactobacillus , while all three studies identified Staphylococcus 62. Both Ruiz et al.31 and62 also reported Escherichia spp. to be prevalent. Lactococcus, the most abundant genera identified in this study, is also commonly found in human colostrum30, and antibodies against Lactococcus phages have previously been noted in cow colostrum65.Likewise, the colostrum microbiota determined in this study were similar to those previously reported in cattle42) may indicate that the sample also reflects allocthonous microbiota picked up from the environment in addition to any autochthonous skin microbiota. We do, however, note that the major genera identified on the udder skin in this study are well documented among skin microbiota of cattle61 and other mammalian species66 where they have been shown to exhibit temporal stability66. Though it is noteworthy that many are dually observed on skin and in the environment (e.g. soil and water), including Arthrobacter, Pseudomonas67, Streptococcus68, Acinetobacter70, and Corynebacterium and may be passed between host and environmental niches72. It is therefore plausible that many or all of the microbes shared between the udder skin and calf GIT were not maternally transferred but instead seeded directly or indirectly, perhaps even continuously from the environment.The entire calf GIT microbiota appeared to be influenced by microbes of the dam’s vagina, udder skin, and colostrum. All three maternal sources comprised a unique microbial reservoir that shared OTUs with all examined calf GIT locations and in both luminal and mucosal subsamples. These shared OTUs were detected in the earliest samples and persisted through the 21 day sampling period. The udder skin shared the greatest number of OTUs with almost all regions of the calf GIT at each time point. Because calves had no contact with the dam following their separation at birth, when these samples were collected, this relationship between the GIT and udder skin microbiota cannot be reflective of microbes transferred in the opposing direction from the calves oral microbiota to the teat and surrounding skin surface. However, the finding that the udder skin exhibited little deviation from the assumptions of neutral theory  do not support this as a typical route of transfer74. Nevertheless, the presence of these rumen microbes in the vagina avails an early opportunity to seed the neonatal calf GIT and understanding this relationship may be important to on-going ecological engineering efforts aimed at replacing ruminal methanogenesis with alternative forms of hydrogenotrophy75.This is less likely to be true of vaginal or colostrum microbes. The vaginal microbiota are likely to represent the first source of microbes due to the order in which the calf is exposed to each of these maternal sources. Despite the brevity of this interaction, the vagina appears to have a profound influence contributing many microbial taxa with well-described physiological roles in the adult rumen. This includes a nearly unique reservoir of methanogenic archaea among the maternal sources profiled in this study. Methanogenic archaea, alike many of the bacterial genera observed in the vagina are strict anaerobes61, supporting the hypothesis that colostrum possess an autochthonous microbiota. Many of the microbes we detected in colostrum were also observed in the calf GIT, suggesting colostrum contributed to the make-up of the calf GIT. Colostrum is known to be of additional importance to host physiology and microbial ecology because it provides the earliest form of nutrition both directly to the neonate and to the early microbiota76, along with immunological factors77. Consequently, the timely acquisition of high-quality colostrum is a proven factor in sustaining the GIT health of young calves77 and it would be interesting to more explicitly examine the nutritive and immunological influences of these colostrum-mediated factors on the early ruminant microbiota.Colostrum exhibited the largest deviation from the assumptions of neutral theory and similar to previous reports, was found to possess a unique microbiota from that observed on the surrounding skin78, which could facilitate the oral transfer of important rumen microbial groups, similar to those observed in the dam’s vagina. However, because calves were separated from the dam within 15 minutes of parturition, the direct transfer of maternal microbes from the oral cavity was limited. Feed-borne microbes may also be important, although a study by Piao and colleagues79 revealed a rapid loss of signal for microbes colonizing switchgrass upon immersion within the rumen. It is also unclear if calves are colonized in utero by a placental or uterine microbiota. Studies have recently revealed microbial signatures in the ruminant uterus81 and it would be interesting to establish if these microbes are viably transferred to the calf prior to parturition.These were clearly not the only sources of microbes to the neonatal calf GIT as evidenced by the finding that only ~46% of OTUs observed in the calf GIT were detected in one or other maternal sample. Under ordinary circumstances, the dam’s oral microbiota may also be important. This is particularly true given cattle frequently regurgitate feed from their rumen in a process known as rumination16 and dairy cattle15 also found no significant variation between the colon and either feces, or the rectum, respectively. The additional relationship with the abomasum observed in this study may reflect early ruminant GIT physiology where a unique evolutionary feature, the oesophageal groove, that causes ingested feed to bypass the underdeveloped rumino-reticulum and instead empty directly into the abomasum82. Perhaps in relation to this, Firmicutes were seen to have significantly greater relative abundances in the colon and conversely Bacteroidetes had significantly greater relative abundances within the bypassed foregut. This delineation is reflective of observations of various animals during and after hibernation where the relative abundance of Bacteroidetes in the GIT has been seen to be greater than Firmicutes when the animal is hibernating, while Firmicutes rapidly outnumber Bacteroidetes following emergence and a return to eating84. The ratio of Bacteroidetes to Firmicutes could therefore reasonably be hypothesized to reflect the differing nutritional status along the GIT. It is also worth noting that the greater relative abundance of Bacteroidetes in the rumen is inconsistent with studies of the matured adult rumen where Firmicutes are typically shown to have the highest relative abundances in both the rumen and colon86. Within each GIT location, differences were also seen between luminal and mucosal samples likely reflecting the differing nutritional and physiological conditions in these regions. For example, genera well described as aerobic or facultatively anaerobic including Escherichia, Pseudomonas, Acinetobacter, and Delftia were seen to have the greatest relative abundances in many of the mucosal samples and this may reflect their resistance to, need of, or preference for higher oxygen concentrations available nearer the gastrointestinal epithelium24. This may also explain why udder skin microbiota almost always exhibited the strongest relationship to the mucosa. As has been described in other studies of microbiota succession, we observed increasing richness and α-diversity with age, and show that this is observable throughout the GIT with the exception of the distal jejunum.Despite little measurable deviation from the assumptions of neutral theory in the calf GIT microbiota, it was found to exhibit biospatial and longitudinal differences over the first 21 days. Only several anatomically connected regions were determined to be inseparable by measures of β-diversity, along with the abomasum, which was seen to be similar to the colon and feces. Two previous studies reporting biospatial variation through the GIT of lambsCollectively, we have provided the first description of the maternal influence on the early ecological dynamics of microbiota throughout the calf GIT, revealing immediate biogeographical delineation of these microbiota among each GIT location and between mucosal and luminal regions. We have also uncovered unique and potentially important influences of microbiota from the skin of the dam’s udder, the vagina, and colostrum, which each appear to make early contributions to the biospatial and longitudinal succession of microbes throughout the early calf GIT.All experimental procedures were performed at Fair Oaks Farms  and University of Illinois Veterinary Medicine Research Farm  under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Illinois (protocol #13096).All authors have read and approved the final version of the manuscript and consent to its publication.Raw sequencing data and metadata are available through NCBI under BioProject accession PRJNA314799.Dataset 1


Tvrm4/Rho+ rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of Rho Among them, individuals suffering from Retinitis Pigmentosa (RP) share a phenotypically broad correspondence characterized by a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision. Subsequently also daylight vision and visual acuity are impaired, ending up with legal blindness. RP is a family of disorders typically caused by a single mutation in any one of numerous, often functionally distant genes, 60 of which have been identified so far. They may encode for a variety of proteins and among them: visual cycle or phototransduction enzymes, ciliary traffic proteins, transport and cytoskeleton components, ion channels, transcription factors, nuclear receptors1. Most of the genes involved in RP are specific for photoreceptors, or the retinal pigment epithelium (RPE), or the retina; RP mutations can be transmitted according to autosomal dominant, recessive, X-linked and mitochondrial patterns of inheritance. Thus, defining RP as a highly genetic heterogeneous disease is justified by the large number of underlying mutations, the diverse functions of mutated genes and the variable modality of inheritance, notwithstanding the occurrence of a fairly homogeneous phenotype.A large variety of experimental approaches aimed at sight restoration or prevention of vision loss caused by retinal degeneration have been recently proposed. Irreversible photoreceptor diseases can arise from hundreds different gene mutations  of RHO. Class I mutations mostly occur near the C-terminal of the protein, which still retains the ability to bind to 11-cis-retinal and to form a functional chromophore but may not be properly transported to the outer segment and shows constitutive activation or increased transducin activation4. The more common class II mutations occur in the transmembrane or cytoplasmic domains of the protein, resulting in rhodopsin misfolding, retention in the endoplasmic reticulum and failure to bind to 11-cis-retinal.Some 40% of Retinitis Pigmentosa cases display autosomal dominant inheritance, and 25–30% of these are attributable to mutations in 6. Such mutations are particularly challenging for gene therapy approaches, as they require suppression of the expression of the native gene and gene augmentation at the same time7. Clearly, rhodopsin stability and efficiency tolerates only few aminoacid changes, as reflected in the finding that the protein shares more than 95% sequence identity even in different species, like mice and humans. Rhodopsin expression, synthesis, folding, translocation to the outer segments of rods and turnover are highly regulated processes, whose impairment can easily lead to cell death, also considering that the photopigment contributes some 90% of the whole protein content of outer segment membranes.Dominant mutations might be associated to a gain-of-function of the endproduct, as shown for the P23H class II mutation, where rhodopsin retention in the ER is followed by a continuous unfolded protein response (UPR), inhibition of the proteasome and aggregation in high molecular weight oligomers, ultimately forming toxic inclusion bodiesRHO mutations are of key importance for understanding the complex link between primary genetic defect and cell death, and for developing appropriate approaches for vision preservation in humans with identical or similar mutations. Yet, available RHO models are few compared to the number of known mutations in the rhodopsin gene: P23H mice and rats mimic the most common human mutation of RHO and have been exploited extensively; however, some strains manifest their phenotypes relatively late in life and with a too slow progression for efficient testing of experimental treatments; moreover, P23H mice are transgenics8 like T17M, P347S9 and S334X mice  originally described by12, and constituting a model of autosomal dominant RP where a missense mutation of RHO changes amino acid 307, isoleucine (ATC), to asparagine (AAC). Ile-307 is in the 7th transmembrane region of rhodopsin and is conserved across species, including humans. Peculiar features of Tvrm4 mice are that a) being generated by mutagenesis, they are not transgenics and have normal levels of rhodopsin; and b) they do not show retinal abnormalities if grown in normal ambient light but undergo RP-like retinal degeneration when exposed briefly to strong, white light, which is otherwise innocuous for wild type (wt) controls.Here, we provide the secondary characterization of retinal phenotype of a mouse strain  maintained in normal ambient light is morphologically undistinguishable from that of wild type  littermates and shows no signs of abnormality in general organization and laminar structure Fig. . A norma2, or 32–35% of the retina, which has an average area of 16.0 mm2. At times, the irradiated spot has an irregular shape and is less extended on the temporal quadrant. Inside this visible “black hole”, the ONL thickness has decreased from the normal 10–12 rows of nuclei to approximately 6 rows. Remaining rods have shortened and irregular outer segments .Immunocytochemistry for a cone bipolar cell marker (synaptotagmin) highlights the discrepancy between the morphology of the seriously degenerated outer retina, and that of the inner layers, where the axonal arbors of cone bipolar cells outline a still intact lamination Fig. . In wholTo determine the functional correlates of the morphological findings described above, we performed an extensive retinal functional assay on Ht Tvrm4 mice subjected to three protocols of light-induction  and in which we recorded the flash electroretinogram (ERG) in scotopic and photopic conditions at different times  from phenotype induction.We confirmed that before exposure to bright light pulses, the ERG of Tvrm4 mutants is comparable to that of age-matched wt animals (data not shown). However, a drastic reduction of both scotopic Fig.  and photThe scotopic ERG suppression is similar for all induction protocols used and at all recorded times. The reduction of the scotopic and photopic b-wave depends, however, on the time of exposure to the light source, so that for the longest exposure (3 min) the response is more reduced at longer times from the induction Fig. .Two days after induction, both scotopic and photopic ERG responses start to decline compared to control animals Fig. . After 7Correspondingly, degeneration of the ONL in the central retina area of Tvrm4 mice exposed for 1 min to 12,000 lux is extensive 2 weeks after exposure, when outer segments of rods are greatly decreased in length and density and only few cones can be stained Fig. . Residua12 exposures longer than 1 min cause the formation of large DNA aggregates in the ONL, indicative of the simultaneous degeneration of a large fraction of photoreceptors with consequent condensation of multiple degenerating nuclei, visible in Fig. Exposure of Tvrm4 mice to the same luminance for increasingly longer times  results in affected areas of larger extension and covering the central 38–40% of the retinal surface, as measured using photoreceptor or DNA staining methods. The corresponding pattern of degeneration is morphologically similar to the one described above (i.e. a rod-cone degeneration with center-to-periphery gradient) but more rapid, with disappearance of the ONL in the central retina achieved in less than 7 days with 3 min illumination. As previously shown by16; onset of abnormal discharge properties in ganglion cells17).Interestingly, we found that longer time exposures at a lower luminance  induce a pattern of degeneration covering almost 75% of all the retinal surface and sparing only the far end of the retinal periphery. With this induction protocol, most of the ONL is reduced to 3–4 rows of nuclei one week after light exposure and the photoreceptor degeneration pattern occurs simultaneously over most of the retinal surface. This paradigm of exposure that causes a general photoreceptor decrement and an abrupt decay of scotopic ERG Fig.  could beBecause of an advantageous correspondence between morphological and functional observations and a relatively mild pattern of retinal degeneration, we chose to further extend our analysis using a 12,000 lux, 1 min exposure inducing protocol. The induced, gradual paradigm of phenotypic manifestation seems particularly useful for future rescue studies of photoreceptor degenerations , although one should be aware of the fact that the phenotype is restricted to the central retina.18. Because Tvrm4 mice of the present study are adult, we investigated whether the hallmarks of phase 1 remodeling observed in other forms of retinal degeneration (i.e. rd1 mice), in which phenotype onset overlaps to the late retinal synaptogenesis, also occur in the novel mutant. Morphological and functional data are reported in Supplementary Materials.Alterations affecting inner retinal neurons in the immediate stage accompanying photoreceptor death are known as phase 1 remodeling and mostly involve second order neurons  and retinal gliaMicroglia recruitment and macroglia activation accompany photoreceptor loss in the Tvrm4 mutant. Numerous microglial cells in active, amoeboid form are observed in the superficial part of the ONL since the very initial stage of photoreceptor death. Large (Iba1-positive) elements are seen to engulf the nuclei of dying rods Fig. . In the Retinal macroglia also activates in parallel to photoreceptor death: as early as 48 hrs post-induction using 1 min, 12,000 lux exposure, Muller cells show the expected upregulation of GFAP immunoreactivity Fig.  throughoThe analysis of the phenotype of Tvrm4 mice exposed for 1 min to 12,000 lux was followed up to 30 days after light-induction. At this time, microscopical analysis shows that the average size of the central degenerated zone did not expand compared to the average size measured at shorter times after induction Fig. . One monP = 0.63. Conversely, photopic ERG recordings show that the functionality of the cone pathway remained unvaried throughout the same period of time  in which the mutation becomes expressed during the first 10 days of life, when photoreceptors undergo the final stages of maturation and synaptogenesis. The possibility to study photoreceptor death and neuronal remodeling bypassing the stages of retinal development and synapse formation is important because in humans, RP is a disease of adulthood, with symptoms that rarely overlap with retinal development. In addition, the overlay of photoreceptor degeneration and retinal development makes it difficult to distinguish between primary effects of the underlying mutation and retinal reorganization due to interactions of photoreceptor death and development completion22.This study provides the secondary characterization of retinal phenotype of Tvrm4 mice, which carry a dominant 22 and to other mutants23, therefore we conclude that these are primary, pathognomonic signs of RP, independently from the age of onset of the phenotype and from the causative mutation.Here, we find that pattern of photoreceptor death, most relevant aspects of phase 1 remodeling, and corresponding ERG signature, are all very similar to those already described in rd1 and rd10 mice during the first month of lifeFuture studies comparing the results obtained here with a phenotype assessment in Tvrm4 immature mice will provide valuable information on the consequences of the Tvrm4 rhodopsin defect when combined with photoreceptor immaturity.RHO models of RP, Tvrm4 mice are not transgenics and therefore exhibit normal levels of rhodopsin. This is important in view of the notion that levels of rhodopsin in photoreceptors are tightly regulated and higher than normal amounts of this protein are believed to be toxic per se for the normal retina24. In Tvrm4 mutants, any abnormality observed upon light induction can be attributed to the sole effect of the mutation.Unlike other 25.Tvrm4 mice exhibit the typical sensitivity to light of animals and humans with Retinitis Pigmentosa; numerous retinal degeneration mice have been shown to undergo faster retinal structural and functional decay if exposed to higher environmental light levels; conversely, photoreceptor degeneration slows down in dark rearing conditionsRHO27, for their typical delay in dark adaptation and slow recovery after photobleaching, described for Tvrm4 mutants in the original paper by12. Together with normal retinal structure and rhodopsin expression, defects in dark adaptation and a highly localized pattern of initiation of photoreceptor degeneration are hallmarks of Tvrm4 mutants, English Mastiff dogs and RP patients with so-called class B1 RHO mutations28. Noticeably, T4R dogs are extremely sensitive to environmental light, and even moderate illumination levels, as used in routine ophthalmological examination, accelerate retinal degeneration.More specifically, Tvrm4 mice recall English Mastiff dogs with a T4R dominant mutation of As for the T4R dogs, we used heterozygous Tvrm4 mice for our study, to better mimic the genetics of dominant human mutations of Rho; however, the homozygous individuals we examined did not show any visible difference from the heterozygous in terms of severity of the induced phenotype and pattern of retinal degeneration.RHO mutation to rod demise but this is indeed an important topic to study, given the relevance that RHO genetic defects play in human RP.The phenotype induced in Tvrm4 mice by strong light is likely triggered by accumulation of large amounts of bleached mutant rhodopsin, which shows a gain of function ultimately leading to photoreceptor death. We made no attempts to trace the cellular pathways connecting the Tvrm4 30) can be adequately studied; the adjacent, virtually intact retinal area can serve an internal control for a number of functional, biochemical and molecular biology investigations.In these studies, one could also investigate the very interesting topic of non-cell-autonomous signaling among retinal cells. Noteworthy is the fact that the death signals involving i.e. photoreceptors, activated macro and microglia, blood vessels, located at the interface between illuminated and dark retinal areas, remain confined to a central spot and do not affect the survival of adjacent cells. The Tvrm4 pattern of degeneration is a typical rod-cone decay in which non-cell-autonomous mechanisms of survival and death  to test accurately light stimuli acting as irreversible or reversible conditions for rod survival and outer segment genesis in an adult mammalian with a rhodopsin dominant mutation, thus opening the path to cell biology studies of great value for photoreceptor biology and repair.Most likely, partially damaged rods at the border between severely damaged and intact area can recover, possibly when the bleached rhodopsin accumulated in the outer segments has been cleared and new discs, with freshly synthetized protein, are generated at the outer segment base. The process of synthesis of new discs is known to take place at a rate of about 2 µm/day in mouse rods32, monocular pharmacological treatments followed by morphological and ERG assays33, retinal/specific gene expression, studies of the effects of photoreceptor degeneration on cortical plasticity34 etc.A further advantage of the model is represented by the possibility of exposing to the inducing light one eye only. Dilating only one pupil completely prevents induction of the phenotype in the other retina, thus providing a useful control when simultaneous testing of the two eyes is desirable. Examples include monocular gene therapy35.In view of the very different areas that are illuminated with different exposing protocols, we conclude that a) short time exposure are recommended for morphological studies in which wild type, non-induced areas can be unequivocally identified by photoreceptor staining methods; these protocols are very useful when cell interactions occurring at the interface between intact and degenerating tissues are being studied. An example is represented by local factors affecting the survival of rods and cones. b) medium time exposures (2 minutes) are optimal for the combination of morphological, biochemical and mass-electrophysiology responses and for possible rescue studies; c) long time exposures (5 minutes), at lower intensity, are ideal for ablating a large population of photoreceptors simultaneously and to study structural and functional effects on the inner retina , for instance using electrical imaging methods to reveal the activity of bipolar cellsWhen a short time exposure  is used, a nasal-temporal asymmetry in the pattern of degeneration may occur in some animals. This has to be taken into account during the experimental design; however, the effective pattern of induction can be visualized easily in retinal whole mounts stained post-mortem.36; or calcium imaging37). Compared to commonly used phosphodiesterase mutants, Tvrm4 mice recall rd10 mice insofar complete photoreceptor degeneration occurs within about one month from phenotype onset19 making functional studies possible; yet, the initial phase of rod death is quite fast in the rhodopsin mutant studied here and more reminiscent of photoreceptor death onset in the aggressive rd1 phenotype22. However, the Tvrm4 degeneration turns into retinal degeneration only as a consequence of interaction of light and rhodopsin and the phenotype might affect only part of the retina. This explains the persistency of an ERG b wave even at the latest times studied here, unlike what observed in rd1 and rd10 mutants, in which the ERG extinguishes after about 2 and 9 weeks of life, when rods and cones have degenerated completely.Overall, the degeneration pattern triggered in the Tvrm4 is rather acute  but still manageable for performing structural and functional analysis; moreover, the spatial extent of retinal degeneration can be modulated by changing the illumination conditions, i.e. longer time exposures at lower intensities lead to larger areas of retinal degenerations. This is likely due to the fact that the mouse has a highly-curved eye, which makes it difficult to illuminate the retina completely, in a very short time, in awake and moving animals. Longer time exposures increase the probability of the light to reach and trigger the phenotype in peripheral zones of the eye; this experimental paradigm might be chosen when recording responses from large retinal areas  and by the Ethical Committees of both Institutions. Protocols adhere to the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in research.Tvrm4/Rho+) with a I307N (near C-terminus) mutation of the rhodopsin gene (Rho) and C57Bl6J wild type  mice were used for this study12. Tvrm4 mutants are on a C57Bl6/J background and exhibit no retinal phenotype unless exposed to bright light stimuli. Genotyping was performed following12 and Jackson laboratories indications.Heterozygous (Ht) Tvrm4 mice , 12,000 lux exposure; they were examined at 48 hr, 7,14, 21 and 30 days post induction, using for a minimum of 3 and a maximum of 8 Ht mice per group.Fourteen additional Ht Tvrm4 mice were used for retinal histology following 2 min, 12,000 lux exposure. A minimum of 3 and a maximum of 5 mice per group were used for studies at 48 hr, 7, 14 and 21 days post induction. N = 3 animals were used for histological studies at 48 hr post induction; n = 8 mice for studies after 7 days. A total of 5 wt mice were used for controls.A total of 3 Ht and 2 wt mice were used for retinal histology after exposure to 12,000 lux inducing light for 5 min and examined 7 days post exposure. Finally, 9 mice were used to study the effects of 6,000 lux exposure for 3, 5, and 10 min. Mice were 3 per group; those exposed for 3 min were harvested after 48 hr; those exposed for 5 and 10 min were harvested after 7 days. In total, 64 Tvrm4 Ht and 14 wt mice were used for histology.Additional homozygous (n = 3) and Ht (n = 6) Tvrm4 mice were used for initial experiments to set up illumination conditions, duration of the inducing light and demonstration of the possibility of inducing unilateral retinal degeneration by dilating only one pupil.Sixty additional Ht Tvrm4 mice  and 6 wt (littermates) were used for ERG recordings after exposure to different inducing light intensities and assessment after various times, as detailed below.ad libitum on a 12/12-hour light/dark cycle, with illumination levels below 60 lux.Ht Tvrm4 mice and wt littermates  were dark adapted for 4 hr, then given 1 µl eye drops of 0.5% atropine (Allergan); after 10 min, mice were placed in a black box  lined with mirrors on the inner sides and closed with a lid holding 3 neon bulbs . Light intensity could be varied by changing the number of illuminated bulbs and was monitored by inserting in the box a light meter sensor ; duration of exposure could be programmed with a digital clock controlling the light bulb switch. Mice were exposed to light pulses of 6,000 or 12,000 lux having durations of 1, 2, 3 or 5 minutes. In the initial experiments, during the light pulses, the temperature of the cage in the illuminating box was monitored with the precision of 1/10 °C using a digital thermometer whose sensor was placed inside the box. No changes were observed with respect to identical measurements done in the absence of the light pulses. Most of the phenotypical analysis refers to a single light exposure of 1 min having an intensity of 12,000 lux. Control animals were a) wild type (wt) littermates of Tvrm4 mice, also exposed to bright light pulses of identical intensity and duration; b) Ht Tvrm4 mice which were not administered atropine; c) Ht Tvrm4 mice in which only one eye was treated with atropine. After bright light exposure, mice were returned to their cages and maintained in a local facility with water and food 39.To verify the possibility of induction of anesthetized animals, 3 Tvrm4 mice were anesthetized with Avertine as above, pupil-dilated, placed in a stereotaxic apparatus, maintaining the animals immobilized and with their eyelids open, and exposed for 2′ at 12.000 lux. One awake, non-restrained Tvrm4 mouse was also exposed to the same inducing protocol. ERG recording was performed after 48 hours on these 4 mice. Only the awake Tvrm4 mutant did develop a retinal degeneration phenotype, while the anesthetized animals remained unaffected, returning minimal ERG decrements. We concluded that an effective induction protocol requires the presence of intact eye movements, probably necessary to maximize the retinal illuminated (and thus degenerated) area. Eye movements are greatly reduced by most anesthetics including AvertineFor retinal histological studies, 2, 7, 14, 21 and 30 days after light induction, mice were deeply anesthetized with intraperitoneal injections of 0.1 ml/5 g body weight Avertin , their eyes quickly enucleated and the animals killed by cervical dislocation. Eyes were labeled on the dorsal pole, the anterior segments removed to obtain eye cups and fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, PB, pH7.4, for 30′ or 1 hour, at room temperature. Eye cups were washed extensively in PB, infiltrated in 30% sucrose in PB, frozen in Tissue-Tek O.C.T. compound  using cold isopenthane and stored at −80 °C until use. Additional whole eyes were also stored using the same protocol. Consecutive cryostat sections (12–14 µm thick) were collected on Super Frost slides and used for immunocytochemistry (ICCH).40, by incubation in a) block solution with 0.3% Triton-X 100, 5% of the serum of the species in which the secondary antibody was generated and 0.01 M Phosphate Buffer Saline (PBS); incubation time was 2 hr for the sections and overnight for whole mounts; b) primary antibody (Ab), diluted in PBS, 0.1% Triton-X 100 and 1% serum; incubation time was overnight for the sections and 3 days for whole mounts; c) fluorescent secondary Ab, diluted as the primary Ab; incubation time was 2–3 hr for the sections and 2 days for whole mounts. Incubations steps longer than 2 hr were done at 4 °C.Contra-lateral eyes (or eyes from different mice) were used to prepare retinal whole mounts, in which the retina was separated from the pigment epithelium and flattened by making four radial cuts toward the head of the optic nerve, maintaining a reference on the dorsal pole. ICCH on both retinal sections and whole mounts was performed followingMouse monoclonal, primary Abs used for retinal sections were: Rhodopsin ; PSD95 ; synaptotagmin 2 ; alpha G0 ; light sensitive channel , used 1:1000); non-Phosphorylated Neurofilament H ; Protein Kinase Cα .Rabbit polyclonal, primary Abs were: S and M/L cone opsins ; cone arrestin ; Calbindin D ; Protein Kinase Cα ; Glial Fibrillary Acidic Protein . A sheep mGluR6 polyclonal antibody was a kind gift of Catherine Morgans . A goat Choline Acetyltransferase  polyclonal was also used. Abs against cone opsins, calbindin D, Iba1 , Neurofilament 200 kDa  and PKCα were also used in whole mount preparations, at 10x the concentrations used for sections.Secondary antibodies were: donkey anti-mouse Alexa Fluor 488 ; donkey anti-rabbit Rhodamine Red X (715296151); donkey anti-goat (705-546-147); and Donkey Anti- Sheep (713-546-147), all from Jackson ImmunoResearch laboratories, USA; these were diluted 1:1,000 for retinal sections and for whole mount preparations. For nuclear counterstaining, retinal sections were incubated for 2 minutes in Hoechst (33342) or in Ethidium homodimer-1 (E1169), both from Life technologies, Italy, diluted 1:1,000. After rinsing in PBS, specimens were mounted in Vectashield  and coverslipped.Images of retinal preparations were obtained with a Zeiss Imager.Z2 microscope equipped with an Apotome2 device , using a Plan Neofluar 40x/1.25 oil objective; or with a Leica TCS-SL confocal microscope equipped with 488 and 453 lasers, using a Plan Apochromat 40x/1.40 oil objective. Images were saved as tiff files; brightness and contrast were adjusted with the Zeiss software ZEN®PRO 2012 or with Adobe Photoshop. Retinal whole mounts were also imaged with the Imager.Z2 microscope using EC Plan-Neofluar 5x/0.16 M27, 10x/0.3 M27 and 20x/0.50 M27 objectives; images were tiled with ZEN module “Tiles & Positions” software to reconstruct the entire retinal surface.Areas of maximum photoreceptor loss were measured on retinal whole mounts stained for cone specific opsins and mounted photoreceptor side-up. Full retinal montages were obtained with Apotome2 using a 20x objective, saved as tiff files and transferred to a Metamorph image analyzer. Images were homogeneously thresholded for brightness and the darkest, central region, surrounding the optic nerve head (ONH), automatically traced and measured. The diameter of the photoreceptor-degenerating zone was also measured on equatorial retinal sections stained with photoreceptor specific antibodies and nuclear dies. Retinal vertical sections  were used to measure the ONL thickness 48 hr, 7, 14 and 30 days after light exposure. A 20x objective and a Zeiss Axiocam color camera were used to take an image per section, centered to the ONH and measuring 600 × 400 micrometers. Rows of nuclei in the ONL were counted semi-manually with Metamorph and the data averaged.42. Scotopic ERG recordings were average responses (n = 5) to flashes of increasing intensity  presented with an inter-stimulus interval ranging from 20 s for dim flashes to 1 min for the brightest flashes. Isolated cone (photopic) components were obtained by superimposing the test flashes (0.016 to 377 cd*s/m2) on a steady background of saturating intensity for rods (30 cd/m2), after at least 15 min from background onset. Amplitude of the a-wave was measured at 7 ms after the onset of light stimulus and the b-wave was measured from the peak of the a-wave to the peak of the b-wave. Oscillatory potentials (OPs) were also measured in both scotopic and photopic conditions. The b wave OPs were extracted digitally by using a fifth-order Butterworth filter as described before44. Peak amplitude of each OP (OP1–OP4) was measured. The ERG data for each condition of light-induction were collected from at least 6 different animals.Animals were anesthetized with 20% Urethane , used at a concentration of 0.1 ml/10 g body weight. ERGs were recorded from dark-adapted mice by means of coiled gold electrodes making contact with the cornea moisturized by a thin layer of gel. Pupils were fully dilated by application of a drop of 1% atropine . Light stimulation and data analysis were as previously described in detailGargini et al. supplementary


Lactobacillus salivarius IBB3154 plus galactooligosaccharide (Syn1) and Lactobacillus plantarum IBB3036 plus raffinose family oligosaccharides (Syn2).In order to discover new strategies to replace antibiotics in the post-antibiotic era in meat-type chicken production, two new synbiotics were tested: (th day of egg incubation (2 mg of prebiotics + 105 cfu bacteria per egg). Hatched roosters  were reared on an experimental farm, kept in pens , with free access to feed and water. After 42 d animals were slaughtered. Blood serum, pancreas, duodenum and duodenum content were collected.The synbiotics were administered via syringe, using a special automatic system, into the egg air chamber of Cobb 500 broiler chicks on the 12P < 0.05). The decreased expression of incretins coincides with changed activity of digestive enzymes in the pancreas and in the duodenal content. The results indicate that incretins are involved in the action of Syn1 and Syn2 or that they may even be their target. No changes were observed in key hormones regulating metabolism  or in metabolic indices . Additionally, synbiotics did not cause significant changes in the activities of alanine and aspartate aminotransferases in broiler chickens. Simultaneously, the activity of alkaline phosphatase and gamma glutamyl transferase diminished after Syn2 and Syn1, respectively.Syn2 increased trypsin activity by 2.5-fold in the pancreas and 1.5-fold in the duodenal content. In the duodenum content, Syn2 resulted in ca 30% elevation in lipase activity and 70% reduction in amylase activity. Syn1 and Syn2 strongly decreased expression of mRNA for GLP-1 and GIP in the duodenum and for GLP-1 receptors in the pancreas. Simultaneously, concentrations of the incretins significantly diminished in the blood serum (The selected synbiotics may be used as in ovo additives for broiler chickens, and Syn2 seems to improve their potential digestive proteolytic and lipolytic ability. Our results suggest that synbiotics can be directly or indirectly involved in incretin secretion and reception. In recent years the administration of natural non-antibiotic and non-hormonal growth stimulators supporting chicken health has been most intensively studied. Prebiotics and probiotics, either alone or in combination (synbiotics), are considered to be a helpful alternative in poultry breeding to replace the usage of antibiotics as growth promoters banned in some parts of the world, strengthen health, improve production parameters and diminish environmental pollution. Both the manner and moment of addition of biologically active compounds seem to be important to achieve the best effects. The in ovo technique applied in the present paper is a relatively new method of supplementation, which allows for the application of prebiotics or synbiotics in early developmental stages and enables the modulation of gastrointestinal tract activity , 2. Besi2, and both derive from the proglucagon molecule by post-translational processing ; Syn2 – Lactobacillus plantarum IBB3036 plus raffinose family oligosaccharides [RFO]) and, besides the enzymatic activity of the pancreas, analyses also focused on enzymatic activity in the duodenum content. Moreover, a wide panel of hormones regulating metabolism , as well as a broad spectrum of biochemical indices  and diagnostic enzymes  were analyzed. All the analyses allowed us to answer the question of whether the chosen in ovo implemented synbiotics act long-term and whether they modulate the hormonal and enzymatic activities of the digestive tract.Considering that the final action of hormones is the result of their synthesis, secretion and reception, the present paper studied the expression of mRNA for GIP and GLP-1 in the duodenum, for their receptors in the pancreas, and the levels of both incretins in the blood serum. Previously, we have demonstrated the effect on the enzymatic activity of the pancreas of in ovo administered synbiotics composed of inulin plus This study was undertaken with the approval of the Polish Local Ethical Commission .Lactobacillus salivarius IBB3154 plus galactooligosaccharide ; Syn2 – Lactobacillus plantarum IBB3036 plus raffinose family oligosaccharides   , 27. TheFor further investigations only those roosters were qualified which had hatched from eggs previously in ovo injected into air chamber eggs . The roosters were reared on a commercial farm registered also as an experimental farm . Birds from control and experimental groups were kept on the floor in pens  and had free access to feed and water. Body weight, feed consumption, feed conversion efficiency and mortality were measured. The rearing data from the experiment are described by Dunislawska et al. . For theOn d 42 birds were slaughtered, the blood was collected and then the blood serum obtained. Immediately after the blood collection, pancreases and duodenum were excised. Also, the duodenum content was taken for analyses. Next, the whole material was frozen and kept for further investigations at −80 °C.The activity of pancreatic enzymes was measured as described previously .g for 16 min.). Activities of amylase, lipase, and trypsin were measured using appropriate commercial colorimetric tests . For measurement of lipase activity, homogenates were diluted 100× using TBS buffer, while for measurement of amylase activity these were diluted 1,000X with commercially supplied buffer. In the case of trypsin measurement, diluted supernatants (100× with TBS) were incubated with 1% enterokinase . The temperatures for measurement of enzyme activities were as follows: for amylase and trypsin 25 °C, and for lipase 37 °C. Activities were measured as the amount of product formed during reactions  and these are expressed in the manuscript as % of control.In brief, after being weighed pancreases were homogenized in TBS buffer on ice and 20% homogenates were centrifuged . The activity of enzymes was measured using the commercial kits mentioned above.To determine the activity of the lipase, amylase and trypsin in the duodenal content, 250 μL of PBS was added to 100 mg of content, the mixture was homogenized on ice and centrifuged , total thyroxine (Thyroxine (T4) kit, RIA – Cis International), free thyroxine (Free Thyroxine (fT4) kit, RIA – Cis International), total triiodothyronine (Triiodothyronine (T3) kit, RIA – Cis International), free triiodothyronine (Free Triiodothyronine (fT3) kit, RIA – Cis International), corticosterone , leptin , incretins GIP (Gastric Inhibitory Polypeptide) , and GLP-1 (Glucagon-like Polypeptide-1) . In order to verify the specificity of binding of kits, serum dilution curve was performed. For this purpose serum from five randomly chickens was diluted 2×, 4× and 10 times in Elisa/RIA buffer, and next concentrations of investigated hormones were determined in all dilutions.The activity of diagnostic enzymes and concentration of main blood biochemical parameters were analyzed in the blood serum. The activities of alanine  and aspartate  aminotransferases as well as alkaline phosphatase (ALP) and gamma glutamyl transpeptidase (GGT) were estimated using commercial kits .The parameters of carbohydrate (glucose), protein , and lipid  metabolism were estimated: glucose was measured using a Glucose Assay Kit – Pointe Scientific (USA), total protein and albumins were estimated using a Total Proteins Kit  and Total Albumins Kit , triglycerides were measured using a kit from Pointe Scientific (USA) , free fatty acids were estimated with a kit from Wako Chemicals (USA), and cholesterol was measured with a Cholesterol/Cholesteryl Ester Quantitation Kit – BioVision (USA).GIP; GLP-1) and their receptors  mRNA and incretins serum concentration is described below. Isolation of total RNA was performed using Tripure reagent  according to the manufacturer’s instructions. The efficiency of isolation and the quality of isolated RNA were determined using NanoDrop 1000. Additionally, the integrity of RNA was determined by electrophoresis in 1% agarose gel. cDNA was synthesized from 1 μg of total RNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Moreover, to exclude contamination of genomic DNA, we performed all RT-PCRs in parallel without added RT and detected no signals. Primers for reactions were designed using Primer-BLAST  and P < 0.05 was considered statistically significant. Statistical significance compared to controls was marked * P < 0.05, ** P < 0.01.Statistical analyses were performed as previously described . In brieGLP-1 mRNA level  and Syn2 (P < 0.05). Similar results were obtained for GIP. Syn1 (P < 0.01) and Syn2 (P < 0.01) decreased its mRNA expression . Statistically significant differences were not observed for GIP-R mRNA in the pancreas  was more pronounced; however, statistically significant changes were also observed in GIP levels (P < 0.05)  Fig. .Fig. 1GLThe decreased expression of incretins coincided with changed activity of digestive enzymes in the pancreas and in the duodenal content. The activity of all investigated enzymes in the pancreas was elevated; however, a statistically significant difference was observed only for trypsin activity in birds which had been administered Syn2. On the other hand, in the duodenal content activity of trypsin and lipase were noticeably heightened after synbiotics with significant changes observed after Syn2. In turn, amylase activity in the duodenum was less than half that of the control value and again a statistically significant change was observed only for Syn2. All alterations are included in Table The concentrations of other blood parameters including hormones are presented in Table Lactococcus lactis spp. lactis; Bi2tos + Lactococcus lactis subsp. cremoris) elevated the amylolytic and lipolytic activity of the pancreas at the end of the rearing period, and synbiotics based on Bi2tos also elevated trypsin activity. In the present paper, we confirmed that synbiotics injected early during development may exert long-lasting effects on pancreatic enzymes. Additionally, the present study also covers the activity of the main pancreatic enzymes in the duodenal content, which reflects their ability to digest three main groups of nutrients: proteins, lipids and carbohydrates. Elevated activities of trypsin and lipase indicate that Syn2 may improve the process of protein and lipid digestion. However, the amylolytic activity in the intestine was strongly decreased after synbiotic treatment. This phenomenon was probably the result of a lower level of secretion of amylase to the duodenum. It is possible that, as a result, the concentration of glucose in the duodenum content was lowered. In comparison to control birds, appropriate averages were 88.7% and 90.4% after treatment with Syn1 and Syn2, respectively; both changes were statistically significant at P < 0.05. This, in turn, was reflected in a lower secretion of incretins to the blood. This could offer an explanation for the parallel decrease in amylase activity in the gastrointestinal tract, diminished expression of mRNA for incretins and their lowered concentration in the blood serum. Additionally, the increase in trypsin and lipase activity could be connected with the lower levels of GLP-1 after synbiotic application. Intravenous infusion of GLP-1 reduced trypsin and lipase activity in men  and Lb. plantarum IBB3036 plus raffinose family oligosaccharides [RFO]), it seems to be obvious that they may be used as in ovo additives in chickens without negative, up to the 6th week, effects on health and basic production parameters. Simultaneously, they must affect the pancreas, evoking significant alterations in the activity of gastrointestinal tract digestive enzymes and in the expression of incretins. So, they influence an important function of the organism, which is proper food digestion, and they downregulate (control) expression of incretins. As mentioned above, synbiotics and prebiotics can modulate intestinal microflora. Based on these findings, literature data indicate a few possible pathways by which probiotics and synbiotics can affect secretion and expression of incretins in K- and L-cells. The most convincing seems to be the effect of microbiomes on short chain fatty acid (SCFA) concentrations in the intestine. SCFA are produced during bacterial fermentation of dietary fiber or of non-absorbed carbohydrate. It was shown in the literature that there exists a link between fiber, gut microbiota and K, L-cells acting via GPR41, GPR43, FFAR2 and FFAR3. Moreover, a possible role of FFAR2 in GLP-1 secretion was suggested by the discoveries that SCFA temporary triggered Ca2+ in primary L-cells and mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro  leads to a lowering of the level of incretins (GLP-1 and GIP) in blood serum. Furthermore, these two synbiotics downregulated GLP-1 and GIP mRNA expression in the duodenum and GLP-1R in the pancreas. Moreover, Syn2 increased trypsin and lipase activities in the duodenum content, simultaneously decreasing amylase activity. Such activity can promote digestion of proteins and lipids. Our results suggest that synbiotics can be directly or indirectly involved in incretin secretion and reception.In summary, we found that in ovo injection of two different synbiotics [


However, the detailed inhibitory role of glycyrrhizin in the growth of lung cancer is still unclear. In this study, we first found that glycyrrhizin inhibited the growth of lung tumor in PDX mice. And high level of HMGB1 promoted the migration and invasion of lung cancer cells, which was suppressed by glycyrrhizin. Moreover, glycyrrhizin reduced the activity of JAK/STAT signaling pathway, which is the upstream regulator of HMGB1. Therefore, this study revealed a potential mechanism by which glycyrrhizin can inhibit the progression of lung cancer.Lung cancer has very high mortality and glycyrrhizin was found to significantly inhibit the growth of lung cancer cells Lung cancer is highly prevalent worldwide. An estimated 526,510 men and women living in the United States had a history of lung cancer in 2016 [ Glycyrrhiza glabra. It was first identified as an antiviral drug [Glycyrrhizin, a glycoconjugated triterpene, is extracted from the roots of licorice plant,ral drug  and has ral drug , 5. Receral drug . Furtherral drug , 7. TherHigh Mobility Group Box 1 (HMGB1) is a conserved nonhistone chromosomal protein that regulates nucleosome formation and gene transcription. HMGB1 could also be released into extracellular matrix, where it is recognized as a cytokine based on its role in mediating systemic inflammatory response . FurtherGlycyrrhizin was found to inhibit HMGB1 for diagnosing HMGB1-mediated lethal systemic inflammation . GlycyrrIn this study, glycyrrhizin was found to suppress the tumor growth of NSCLC in PDX mice. Furthermore, the levels of both HMGB1 and its related JAK/STAT3 signal pathway factors were downregulated by treating PDX mice with glycyrrhizin. These findings indicated that glycyrrhizin may be involved in anticancer therapy of NSCLC through downregulating the level of HMGB1.Human NSCLC cell line HCC827 was purchased from the cell bank of Chinese Academy of Sciences and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS).7 ml−1. Then, 0.2 ml cell suspension was subcutaneously injected into the back of nude mice, and the tumor formation was visually observed after 5 d. When the tumor grew to a diameter of 5–7 mm, 30 mice were randomly divided into the model group and glycyrrhizic acid treatment group, with 15 rats/group.The logarithmic growth phase of HCC827 cells was suspended in cell culture medium and the concentration was adjusted to 1 × 10Glycyrrhizic acid  was diluted in DMSO and intraperitoneally injected at a dose of 100 mg/kg for two days, followed by continuous administration for two weeks. The model control group was injected with the same amount of DMSO.Lung cancer cells from the glycyrrhizin treated and DMSO treated groups were stained by Hematoxylin and Eosin (H&E), as previously described . First, The immunohistochemical staining was performed using EnvisionTM, as previously described . Lung caβ-Actin was used as an internal control. Finally, the membranes were treated with enhanced chemiluminescent system for visualization of the protein bands. The bands were quantified using Image J software.Western blot analysis was performed as previously described . Cells wFor testing the effect of glycyrrhizin on the growth of NSCLC, we established the PDX mouse model using the lung cancer HCC827 cell line. Then, we observed the morphology of the cancer cells in PDX mice and found that the arrangement of cells and shape of nuclei were irregular .p < 0.01)  . These rp < 0.05)  . We also < 0.05) , suggestIn mammals, the JAK/STAT pathway is the principal signaling mechanism of inflammation and involves various cytokines and growth factors , 33. MemWe further examined whether glycyrrhizin could inhibit the activity of JAK/STAT signaling pathway. The phosphorylation status of Jak2 and Stat3 was detected by specific phosphorylated antibodies, which showed that the phosphorylation levels of Jak2 and Stat3 were significantly higher in the PDX-model mice, but obviously lower after glycyrrhizin treatment Figures . These r in vitro and in PDX mice. Subsequently, Deng et al. [In this study, glycyrrhizin was shown to suppress the growth of lung tumor tissues in PDX mice, derived from NSCLC HCC827 cell line, which is consistent with recent reports on the anticancer effect of glycyrrhizin on lung cancer progression , 7. HuanHMGB1, a cytokine, has extracellular functions in inflammation and cancer progression. As a late modulator of inflammation, HMGB1 functions as a damage-associated molecular pattern in the sterile inflammation model by amplifying hepatic ischemia/reperfusion (I/R) and acetaminophen-induced liver necrotic injury , 38. GlyGlycyrrhizin influenced the upstream regulator of HMGB1, the JAK/STAT signaling pathway. HMGB1 release from nucleus to cytoplasm is regulated by the activation of JAK/STAT signaling pathway . We founGlycyrrhizin inhibits the growth of lung tumors in PDX mice by downregulating the level of HMGB1. This mechanism is potentially due to the inhibition of JAK/STAT signaling pathway by glycyrrhizin. Glycyrrhizin may be further investigated as a potential drug for NSCLC.


Pneumocystis jirovecii (PJ) pneumonia ordinarily requires invasive procedures that could be avoided by PCR methodologies, if these could be designed with adequate cut-off values for confounding background carriage.Diagnosis of We designed a novel quantitative real-time PCR assay to detect the mitochondrial large subunit rRNA gene of PJ in oral washes. To benchmark levels of PJ carriage versus infection, we tested asymptomatic immunosuppressed patients including Danish (n = 88) and West African HIV-infected (n = 142) patients, renal transplant recipients (n = 51), rheumatologic patients (n = 102), patients with inflammatory bowel diseases (n = 98), and healthy blood donors . The fungal burden in patients with PJ pneumonia  was also investigated.Danish HIV-infected patients (with viremia/low CD4) and recent transplant recipients were at most risk of being carriers , whereas PJ was rarely detected among rheumatologic patients, patients with inflammatory bowel diseases, and untreated West African HIV patients. PJ was not detected among healthy controls. The fungal burden in patients with PCP fell rapidly on treatment.The quantitative PCR method described could conceivably discriminate between carriage and disease, given suitable threshold values for the former, and predict treatment efficacy by measures of the fungal burden in daily oral washes. Pneumocystis jirovecii (PJ) is a well-known cause of severe Pneumocystis pneumonia (PCP) among immunosuppressed patients, particularly those infected with HIV [The opportunistic pathogen with HIV , 2. PCP with HIV –5.Microscopy of broncho alveolar lavage (BAL) fluid is still the mainstay of PJ detection in conjunction with histologic staining or immunofluorescence , 7, howePJ can also be detected in other specimens obtained by non invasive methods such as nasopharyngeal aspirates, induced sputum and oral wash. PCR methods using oral washes have been trailed in an attempt to avoid invasive procedures ,15 givenThe aims of this study were to 1) develop a quantitative PCR assay to be used with oral wash with which to estimate the frequency of carriage in different groups of immunocompromised patients and healthy bloddonors and 2) compare the fungal burden in immunosuppressed patients with and without PCP to establish a cut-off value.HIV infected patients (n = 88) from the outpatient clinic at the department of infectious diseases, Aarhus University Hospital, were included. They were divided into four groups according to viral load (below or above 1000 copies/mL) and CD4 cell count (below or above 200 cells/μL) at inclusion.Newly diagnosed HIV patients (n = 142) from the Hospital National Simão Mendes in Guinea Bissau in West Africa were included at their first consultation. Patients were divided into two groups according to CD4 count (below or above 200 cells/μL).Renal transplant recipients (n = 51) from the outpatient clinic at the department of nephrology, Aarhus University Hospital, were recruited. Twenty-five patients were examined two to six months after transplantation, with 26 patients examined more than a year after transplantation.Rheumatologic patients (n = 102) from the outpatient clinic at the department of rheumatology, Aarhus University Hospital, were included. These patients were divided into four groups defined by the immunosuppressive medication they received; the underlying diagnosis was not considered. The relevant medications were TNF-α inhibitors (n = 27), IL6 inhibitors (n = 25), rituximab (n = 25), and methotrexate (n = 25).Patients with IBD (n = 98) were included from the outpatient clinic at the department of hepatology and gastroenterology, Aarhus University Hospital. These patients were divided into groups based on the immunosuppressive medication received , n = 48).Healthy blood donors (n = 50) from the blood bank, Aarhus University Hospital, were included as non-immunosuppressed controls.Seven patients with verified or suspected PCP were included. Four attended the department of infectious diseases, Hvidovre Hospital, Copenhagen, with the remainder at the department of infectious diseases, Aarhus University Hospital. In addition to the oral wash, BAL fluids were available from three of the seven cases.DNA was extracted from 0.5 mL of oral wash using the MagNA Pure 96 instrument  and Viral Nucleic Acid Large Volume Kit according to the manufacturer’s instruction. Two TaqMan real-time PCR assays targeting PJ genes were used. One assay amplified a 121 bp fragment of the multicopy mitochondrial large subunit rRNA gene (mtSUrRNA) using the primers PjFI and PJRI  and the probe PjPI .The second assay amplified 104 bp of the singlecopy kex-1 gene from PJ using the primers kexF and kexR , as previously described , with a 20, and 7.5 μL of the TaqMan Fast Advanced Master Mix . The TaqMan real-time PCR assay was performed on a LightCycler 480II . Amplification cycles included 10 min at 95°C followed by 45 cycles of 1 sec at 95°C, then 20 sec at 60°C. Triplicates of standards in tenfold serial dilutions from 10−7 to 10−14 were included.The final reaction volume was 15 μL (5 μL extracted DNA + 10 μL mixture). The reaction mixture comprised the probe (0.2 μM), primers (0.2 μM of each), ddH20 and stored at -80°C.As a positive control, a synthesized single stranded oligo of the 121 base pair DNA fragment generated by the PjFI and PjRI primer set  was incl20, was included to exclude contamination as well as a positive control (standard diluted to 10−8 corresponding to 3 x 106 copies) to assess the sensitivity and quality of each PCR run. An internal control was added to all samples to test for the presence of PCR inhibitor. This consisted of primers and a probe for the Phocine herpes virus (PhHV) [A negative control, consisting of ddHs (PhHV) ,18. Inhis (PhHV) . The TaqThe study was reviewed and approved by the Ethical Committee of Region Midt.A written consent was obtainedfrom study participants and stored together with documents related to the study. This procedure was approved by the ethical commitee.Fluorescence data were analyzed using the LightCycler software . Cp values were obtained using the second derivative analysis mode of the LightCycler software. The automated method identifies the crossing point of a sample i.e. where the fluorescence curve of the sample turns sharply upward. This turning point will be a maximum on the second derivative plot of the reaction. The standards were used to generate a standard curve for the quantification of positive patient samples. Statistics and curves were generated with GraphPad Prism version 6.0 .The singlecopy gene kex1 and the multicopy gene mtLSU were selected as candidate target genes, then compared. With Cp values below 35, the PCR efficiency for the mtLSUrna and kex-1 assays were 100,1 and 98,0 respectively. Although these efficiencies were similar, mtLSU was superior with a difference of four Cp values for the same target concentration (i.e. the detection limit for mtLSU would be 16 times lower than for kex-1). Therefore mtLSU was chosen as the target gene. The relation between Cq and gene copies was linear over a range of 7,5 log Cv values of 4 replicates of each dilution of the standard row ranged from 0,2 to 1,3 . Inter-a−8) performed as expected. Inhibition of the PCR reaction occurred with only one patient, suspected to have PCP. The RNase P assay showed only minor variations between samples and standardization was not necessary. Two samples from a blood donor and a renal transplant patient were negative and were excluded.All negative (water) and positive controls  and, in Danish HIV patients, by HIV-RNA level (above or below 1000 copies/mL). All patients from Guinea Bissau were untreated, thus viral load would be above 1000 copies/mL in the vast majority of patients. Demographics are shown in detail in The HIV patients at greatest risk of being carriers had uncontrolled viremia and a low CD4 count (<200). Three of 13 Danish HIV patients with uncontrolled viremia and a low CD4 count were carriers (23%), and three received PCP prophylaxis with sulfamethoxazole-trimetoprim (SMZ-TMP), which most likely had an effect on detection of PJ. Thus, three of ten patients considered at risk for PCP, but without clinical PCP, were carriers (33%). The PCR positive patients comprised a newly diagnosed 36-year-old male with a fungal burden of 1.089 copies. This patient had a viral load of 1.061.834 copies/mL and a CD4 count of 50 cells/μL. Prior to inclusion he presented with mild respiratory symptoms but no history of PCP. He received antiretroviral therapy immediately after admission, and did not progress to clinical PCP. Only one of the patients with a low viral load and a high CD4 count was colonized (3 copies detected). Seventy-percent of non-viremic patients with a CD4 count <200 received SMZ-TMP at the time of inclusion. None of these patients were carriers.Carriage was less frequent among the Africans at risk for PCP. Thus PJ was detected in only three percent of patients considered at risk. Only one of the PCR positive patients had a CD4 count above 200 (3 gene copies). PJ copy numbers between 3 and 524 were detected. All PCR positive patients were non-smokers and infected with HIV type 1.Demographics are shown in detail in Carriage was seen only in patients who were transplanted less than 6 months before an oral wash was obtained, with none seen in patients on PCP prophylaxis (3). Thus four of 24 patients were colonized (16.7%). Three of these 24 patients received PCP prophylaxis. When omitting those patients, 19.5% of patients at risk were carriers. In the oral wash from one PCR positive patient, 59.300 copies were detected. At the time of his inclusion, he had experienced dyspnoea for a couple of weeks. On follow-up, his dyspnea was still present 3 weeks later, at which time immunosuppression was reduced, resulting in the disappearance of his pulmonary symptoms. He did not undergo further diagnostic procedures.None of the patients treated with rituximab or TNF-α inhibitors were and only one (4%) of the patients treated with IL6 inhibitors was carrier (9 copies). He had respiratory symptoms and had also been prescribed 10–12.5 mg prednisolone for three months. Three (12%) of the patients with rheumatoid arthritis treated with methotrexate as monotherapy were carriers and presented with 6, 28 and 14 PJ copies.None of the patients receiving TNF-α inhibitors were carriers, but one patient (2.1%) receiving a 5-ASA preparation was (3 copies). This was a 22-year-old man with no pulmonary complaints, receiving a daily dose of 25 mg prednisolone as a supplementary treatment.PJ was not detected in any of the healthy blood donors.Carriage was most frequently seen in renal transplant patients within the first 6 months after operation and in Danish HIV infected patients with viremia combined with a low CD4 count.A high fungal load above 1000 copies/ml was suggestive of PCP, since two patients with respiratory symptoms improved without specific treatment after initiating antiretroviral therapy or reduction of immunosuppression. Low fungal load, however, did not suggest PCP, and no patients with a PJ copy number below 30 copies/ml developed symptomatic disease on follow up. One African HIV patient with an intermediate fungal load of 524 copies/ml was asymptomatic. Unfortunately follow up data were not available. Thus, copy numbers in in intermediate range, 30–1000 copies/ml may represent a gray zone within which PJ in oral wash cannot be used to predict incipient PCP with the present data.Seven HIV patients with suspected or verified PCP were included. The three patients shown in Data from the four HIV patients that were not followed up with oral washes after initiation of treatment are shown in The quantitative PCR assay amplifying the mtLSUrRNA gene from oral washes, had a high specificity and sensitivity, with a detection range from 3 to 30.000.000 copies. Standardization of the oral samples was not needed given the homogeneity of the oral wash. Nor was inhibition of the PCR reaction a problem using these samples.In the selection of a target gene a single copy gene, kex1, was compared with a multi copy gene, mtLSUrRNA ,20,21, wCarriage with PJ was primarily seen in Danish HIV infected patients . To our knowledge, no previous studies have been performed on oral washes from this population. Fritzsche et al.  reportedIn studies on deep airway specimens from a mixed group of non-HIV immunosuppressed patients  a prevalence of 14–24% , 12 was Interestingly 12% of the patients with rheumatoid arthritis treated with methotrexate without concomitant steroid were colonized. A Japanese study  reportedWe identified a single IBD patient colonized with PJ. This patient was treated with ASA and a moderate dose of prednisolone, whereas no patients on anti-TNF α were colonized. Long et al. reported a hazard ratio of three for IBD patients, with prednisolone being a major risk factor .PCP is normally diagnosed by demonstrating PJ by immunofluorescence or histological stains in material obtained from deep airways by BAL. Qualitative or quantitative PCR, however, is often used, but as mentioned above with some uncertainty, since carriage has been demonstrated in immunocompromised patients and applying a qualitative method a gray zone is often seen, within which it is not possible to differentiate disease from carriage. Furthermore, a study from Valero et al  suggestsSince BAL is an invasive procedure and may be associated with a risk of respiratory deterioration, detection of PJ by a PCR method in material obtained with non-invasive methods such as oral wash is of interest to diagnose PCP. It is, however, important to emphasize that a negative result cannot necessarily rule out PCP in symptomatic patients, opposite a similar finding in BAL. On the other hand, detection of PJ in oral wash suggest active proliferation in the lungs and may support PJ as a possible aethiologic agent. The quantitative PCR test described in this study is potentially a diagnostic tool for PCP provided an appropriate cut off level is chosen. Such a level may vary between different groups of immunocompromised patients. Furthermore choice of gene for amplification will influence results, since amplification of a multicopy gene will increase sensitivity. Thus HIV infected patients are known to present with a higher fungal burden than other groups of immunocompromised patients. Our results suggest that patients with a high fungal load–here defined as more than 1000 PJDNA copies/ml may represent incipient or acute PCP, whereas patients with a low fungal burden (below 30 copies/ml) were asymptomatic and did not develop PCP on follow-up. Our findings must now be confirmed in a larger study in these groups of patients to establish a reasonable cut off value, however, it seems that in patientstreated with TNF-α inhibitors and IL-6 inhibitors, a positive PCR result might be included in a diagnostic strategy.Oral washes from three PCP patients were obtained before treatment and consecutively on the following days. After initiation of PCP treatment, the fungal burden in the oral washes fell rapidly. Thus, ideally, specimens must initially be obtained prior to treatment. Oral washes from other PCP patients were produced after the initiation of treatment, and consequently the fungal burden in these samples was mostly low, with a maximum of 374 copies at day two. Helweg-Larsen et al.  examinedSince PJ still cannot be cultured, attempts have been made to predict resistance using genetic markers but with mixed results . SequentWe were only able to include a small number of Danish HIV infected patients with viremia and a low CD4 count. The prevalence of colonization in this group is thus less certain. Secondly, only a few PCP patients were included, with most patients examined on treatment. This will reduce the copy number rapidly, and hampered our ability to define a realistic cut off value with which to distinguish PJ colonization from PCP.The quantitative PCR assay developed is specific, has a high sensitivity and large dynamic range. Compared to previous studies, we have systematically and directly compared the prevalence of colonization in disparate groups of immunosuppressed patients. A large number of HIV infected patients from Guinea Bissau at risk for PCP were enrolled, giving a more precise estimate of the prevalence of colonization with PJ.In conclusion, we have developed a sensitive quantitative PCR assay with a large dynamic range and applied the method to characterize the level of carriage in different groups of asymptomatic immunosuppressed patients and the dynamics in patients with PCP using oral wash. Carriage was most frequently seen in renal transplanted patients within the first 6 months after operation and in Danish HIV infected patients with viremia combined with low CD4 count, whereas carriage was less frequent among untreated HIV infected patients from Guinea Bissau and patients on immunosuppressive therapy. A fungal load of >1000 copies was suggestive of PCP, whereas a copy number below 30 was seen in asymptomatic patients, who did not develop symptomatic disease on follow up. A grey zone was seen between these 2 extremes suggesting that the method should be used with caution until larger studies have been undertaken.A higher copy number and a rapid drop of PJ were seen in patients diagnosed with PCP. Thus, the method is potentially useful to evaluate response and drug resistance in PCP patients on treatment.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Primary gall bladder melanoma is a rare and controversial entity. So far, only 36 cases are documented in the literature. Metastatic melanoma targeting the gall bladder, however, has been reported to occur in about 15–20% of melanoma patients and is much more common. Based on the case of a 58-year-old woman presenting with multiple melanoma nodules in the gall bladder, we searched in the available literature in PubMed for articles describing a “primary melanoma of the gallbladder” regardless of language used. We detected 33 papers that described 36 cases of primary gall bladder melanoma between 1907 and 2017. From different criteria distinguishing primary and secondary gall bladder melanoma, generally, the following were accepted: (1) exclusion of previous primary melanoma, (2) absence of synchronous involvement of other sites, (3) unicity of the lesion, (4) polypoid or papillary shape of the lesion, and (5) presence of junctional melanocitary component. Review of the 36 published cases revealed that only about one-fourth of them fulfilled all the five criteria. Primary gall bladder melanoma is even rarer than described in the literature, and the question whether this entity really exists remains open.  The existence of a primary melanoma of the gall bladder is controversial, and only about 40 cases are described in the literature so far. Based on an own case with diagnosis of a melanoma in the gall bladder, we were wondering which criteria should be fulfilled for this diagnosis, how they are able to separate this entity from metastatic melanoma, and how these criteria would match with the formerly reported cases as well as with the history of our own patient.A 58-year-old Caucasian woman presented at our institution with immobilizing back pain, known for 4 years but exacerbating for 3 months. The past history included smoking (about 15 pack-years), appendectomy and tonsillectomy as a child, and extensive endometriosis which required surgical excision of sigmoid colon and hysterectomy sixteen years earlier. Conventional X-rays of the thoracic and lumbar spine revealed degenerative alterations only. However, magnetic resonance imaging (MRI) demonstrated multiple lesions in all vertebral bodies with a pathologic fracture in the 8th thoracic vertebral body. Blood count was completely normal. Suspecting bone metastases of a yet unknown primary tumour, a thoracoabdominal CT scan was performed, revealing disseminated small nodules in both lungs (max. 1.0 × 0.5 cm in diameter) and small polypoid intraluminal lesions in the gall bladder infiltrating the liver , the latMacroscopically, the gall bladder specimen was 9 cm long and 3 cm in diameter. In the lumen, a friable broad based papillary nodule in the fundus (5 cm maximum diameter) and two polypoid satellite nodules in the neck (1 cm maximum diameter) were present. In the cystic duct, a small gallstone was identified. Histopathologically, surprisingly, the nodules corresponded to a malignant melanoma. The tumour was almost completely confined to the mucosa  with onl2 daily for 5 days) and monthly infusions of zoledronic acid were installed. Temozolomide was preferred to the former standard of care Dacarbazine, as equal efficacy was demonstrated [As neither BRAF inhibitors nor checkpoint inhibitors were yet available in clinical practice, a palliative chemotherapy with temozolomide , and thrombocytopenia (36 G/l) as a result of progressive bone marrow infiltration. After installation of an analgesic therapy, the patient was transferred to a hospice institution, where she died a few days later.Malignant melanoma in the gall bladder is rare. It usually represents a metastasis from a primary skin or mucosal melanoma and has been reported to occur in 15–20% of melanoma patients . PrimaryWhether primary malignant melanoma of the gall bladder exists is controversial , 37. Obj