Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes  and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop  Argopecten purpuratus is the second most important industry in Chilean mariculture and is primarily concentrated in the north region of the country  algorithm to identify bacterial strains accurately  or whole genome sequencing (WGS) are essential to provide a better understanding of the taxonomic position of the pathogenic curately .Vibrio strain recovered from a massive larval mortality event occurred in a commercial hatchery, to characterize its pathogenic properties and to describe the chronology of the pathology.Despite that efficient rearing techniques for scallop larvae production that have been developed, Chilean commercial hatcheries are currently suffering recurrent episodes of high mortalities of reared larvae, mainly associated with high levels of vibrios . The ideA. purpuratus occurring in a commercial hatchery located in Tongoy Bay in the north of Chile. Triplicate samples of settled dead and moribund larvae were aseptically collected from the bottom of the rearing tank during its water exchange using a sterile glass flask and were transported to the laboratory for immediate processing. Larval samples were centrifuged at 960 g for 2 min using an Eppendorf 5415D centrifuge  and the water excess was discarded. Settled larvae were ground by hand using a sterile glass digester containing 2 mL of sterile physiological saline  to obtain a homogenate according to the method of The pathogenic strain VPAP30 was recovered from a massive mortality event of reared-larvae of the scallop Vibrio species were performed with different tests following 2, and 0.05% KCl, according to the instructions of the manufacturer and the microplate was aerobically incubated in the dark at 20°C for 72 h.Phenotypic characterization to identify 9 bacteria mL-1), and 65 μL of this suspension were added to each cupule. Test strips were incubated for 4 h at 20°C and following incubation, 1 drop of ZYM A  and ZYM B  were added to each cupule. Test strips were read after 5 min and the results were scored using the following classification: 0, negative reaction; 1–2 weak activity; 3–5 strong activity. The assay was performed twice to ensure reproducibility.The enzymatic activities of VPAP30 strain were determined by using the API ZYM system  according to the manufacturer’s guidelines. Briefly, VPAP30 strain was cultured overnight in TSB2, centrifuged at 4,200 g at 4°C and resuspended in a NaCl 0.85% solution  to obtain a turbidity of 5 McFarland , 1 × polymerase reaction buffer, 2 mM MgCl2, and 0.25 pmol μL-1 of each primer. To identify the bacterial strain, amplification of the 16S rRNA gene from positions 28 to 1,492 was performed using the primer pair 27F and 1492R as previously described (ftsZ), glyceraldehyde-3-phosphate dehydrogenase (gapA), gyrase beta subunit (gyrB), rod shape-determining protein (mreB), uridine monophosphate kinase (pyrH), recombinase A (recA), RNA polymerase alpha subunit (rpoA), and topoisomerase I (topA) were used to perform a MLSA. Amplification of these genes was performed as previously described . PCR was performed as described in escribed . The houescribed . PCR mixescribed  or were V. tubiashii, V. bivalvicida, V. crosai, V. orientalis,V. brasiliensis, V. sinaloensis, V. hepatarius, and V. europaeus) allowed us to establish a complete taxonomic study based on comparisons among genome assemblies with the strain VPAP30. ANI calculations were performed according to BLAST (ANIb) algorithm, using JSpeciesWS  using the BLAST+ method  containing 4 mL of 0.22 μm-filter sterilized seawater to obtain a final concentration of 20 larvae mL-1 and were challenged with a final approximate concentration of 8.0 ± 1.0 × 105 CFU mL-1 of the VPAP30 strain. The pathogen V. pectenicida A365 (The pathogenic activity time course for the VPAP30 strain was studied using an ida A365  was incl50) values after 24 and 48 h of exposure, according to 50 was defined as the dose of the VPAP30 strain required to kill 50% of infected scallop larvae. The VPAP30 strain was tested for its pathogenicity in triplicate using 12-well tissue culture plates . Scallop larvae were added to each well of the tissue culture plate containing 4 mL of 0.22 μm filter sterilized seawater at a concentration of 20 larvae mL-1, and the VPAP30 strain was added to the wells to obtain final concentrations of 1.4 ± 0.4 × 102, 1.4 ± 0.4 × 103, 1.4 ± 0.4 × 104, and 1.4 ± 0.4 × 105 CFU ml-1, using six wells per plate for each concentration. The inverted microscope Olympus CKX41  was used to determine the numbers of live and dead larvae at 24 and 48 h post-inoculation. A group of larvae were also inoculated with filtered seawater as negative control.The virulence of the VPAP30 strain was estimated by determining its 50% lethal dose  containing 3.8 mL of microfiltered seawater and then inoculated in triplicate with 0.2 mL of the cell-free supernatant to obtain a final concentration of 4 μg protein mL-1. Larval cultures inoculated with 0.2 mL of PBS were used as controls. Microplates were incubated at 18°C for 48 h in the dark and the proportion of dead larvae was determined at 12, 24, 36, and 48 h using the inverted microscope Olympus model CKX41 . In addition, supernatant samples of VPAP30 and V. pectenicida A365 strains were heated at 125°C for 15 min, and the pathogenic activity of treated supernatants was assayed in triplicate, as previously described.The ECPs produced by the VPAP30 and e method . BrieflyVibrio strain. The pathogenic strain was cultured in TSB2 (Difco) at 20°C for 24 h with shaking at 100 rpm using an orbital shaker . The broth was centrifuged at 5,725 g for 8 min, then the bacterial pellet was resuspended in 10 mL of sterile seawater and the optical density was adjusted to 0.8–1.3 at 610 nm in a spectrophotometer  under aseptic conditions. The 5-DTAF was dissolved in 0.22 μm-filtered PBS (pH 7.4) to obtain a final concentration of 0.5 mg mL-1. A 0.5 mL aliquot of the 5-DTAF solution was added to 9.5 mL of the bacterial suspension, and the mixture was incubated at 20°C for 1 h in total darkness with shaking at 90 rpm. After incubation, the bacterial culture was pelleted by centrifugation  and resuspended in 0.22 μm-filtered seawater and the procedure was repeated until an unstained suspension was observed. Healthy 10 days old scallop larvae of A. purpuratus maintained in 12-well microplates  at a density of 20 larvae mL-1 were inoculated in triplicate with the stained VPAP30 strain to obtain a final concentration of 1 × 105 CFU mL-1 and were observed at 0.5, 1, 4, 6, 12, 18, and 24 h using the Nikon fluorescence microscope Eclipse 50i. Bacterial concentrations were confirmed by a standard dilution plating technique as previously described . Furthermore, the log-rank test was used to compare the survival rates of larval groups not infected, infected with VPAP30 and infected with V. pectenicida A365 using the Kaplan-Meier procedure. All statistical analyses were performed using SigmaStat 3.1 (Systat Software Inc.).Larval survival percentages were transformed to arcsin [square root ] and were compared using one-way ANOVA. When overall differences were significant, All material contaminated with microorganisms, as well as all used bacterial cultures were discarded after sterilization by autoclaving.Vibrio . Furthermore, the 16S rRNA sequence of VPAP30 strain was aligned with reference using the public database of EzBio Cloud1 website and confirmed V. bivalvicida 605T (Accession number LLEI01000012) as the closest relative for the strain VPAP30.Molecular identification of the VPAP30 strain was determined by16S rDNA sequence analysis . The phylogenetic tree constructed from evolutionary distances of 15 representative strains using the NJ algorithm method is shown in ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) to improve the taxonomic resolution  eight housekeeping genes for encoding various functions   .V. bivalvicida 605T yielded 84.2% DDH similarity, clearly higher than the limit (70%) for delineation of prokaryotic species (V. tubiashii ATCC 19109T (31.5% DDH). Overall, the results obtained from the genomic analyses, including MLSA, ANI and DDH, supported the taxonomic affiliation of the strain VPAP30 as V. bivalvicida. Interestingly, the phenotypic profile of the VPAP30 strain is consistent with the characteristics described for V. bivalvicida  higher levels of larval mortality than those produced by the V. pectenicida strain during all the challenge assays. After 24 h of exposure, larval survival of larvae challenged with the VPAP30 strain was 34.3 ± 4.9%, significantly (P < 0.001) lower than that observed in larvae challenged with V. pectenicida A365 (77.8 ± 4.6%) and not challenged larvae (100%). Larval survival at 36 h post-inoculation with the VPAP30 strain was 0%, significantly (P < 0.001) lower than that observed in larvae challenged with V. pectenicida (61.0 ± 3.6%). Not challenged larvae exhibiting a survival of 97.4 ± 1.2% after a period of 48 h. The LD50 for the VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively.Pathogenic activity of the VPAP30 strain was demonstrated by infecting healthy scallop larvae with VPAP30 and ortality . HoweverThe VPAP30 strain produced on challenged scallop larvae, the classical signs of vibriosis affecting mollusc larvae. These signs were identical to those observed in the larval culture suffering a vibriosis outbreak that developed in the commercial hatchery when the VPAP30 strain was recovered. The main clinical symptoms exhibited by larvae infected with the VPAP30 strain were disruption of the velum, ciliary cells detached from the velum and necrosis of the digestive gland tissue . ErraticThe VPAP30 strain was efficiently stained with 5-DTAF and fluorescence was maintained for at least 36 h , permittV. pectenicida A365 strains, they exhibited identical symptoms to those observed during bacterial challenges. At 12 h post-inoculation with the ECPs of VPAP30 and V. pectenicida strains, percentages of larval survival were 88.6 ± 2.7% and 90.3 ± 2.5%, respectively. Survival rates of larvae infected with VPAP30 and V. pectenicida strains remained at levels not significantly different (P < 0.05) until 36 h post-inoculation . However, at 48 h post-inoculation, ECPs of V. pectenicida produced a larval survival of 41.9 ± 5.4%, significantly (P < 0.05) lower than that produced by the ECPs of the VPAP30 strain (59.5 ± 1.7%), whereas larval survival of the control group was 97.4 ± 1.2%  and flat oyster (Ostrea edulis), some of the most important bivalve species reared in the European hatcheries , supporting the potential threat for the shellfish hatcheries in the Atlantic and Pacific coasts.One of the most important problems to define the current curately , 2013. Rtcheries . PresentV. bivalvicida VPAP30 strain in the dead and moribund scallop larvae samples, recovered almost as a pure culture in a non-selective bacteriological medium, the high pathogenic activity on experimentally infected scallop larvae, resemblance of the chronology of the pathology as well as the capacity to produce the same pathological symptoms exhibited by the sampled scallop larvae, strongly support the hypothesis that the mortality event that occurred in the commercial scallop hatchery in Chile was primarily caused by this strain. Notably, the high density and predominance of the colony morphotype of the VPAP30 strain was not observed in agar plates seeded with rearing tank water and swimming larvae samples. Furthermore, in a similar study, the primary pathogenic role of three V. splendidus strains in hatchery-reared scallop larvae was previously demonstrated in a similar manner, fulfilling the Koch’s postulates  during the first 2 h of infection, Vibrio strains were filtered by larval vellum and entered the digestive system through the esophagus and stomach colonizing the digestive gland and intestine; (2) then, Vibrio strains spread and proliferated to the surrounding organs in the body cavity (6–8 h post infection), and (3) after 14 h of challenge, the body cavity was completely colonized by Vibrio strains. Interestingly, DTAF-stained cells of the VPAP30 strain maintained their virulence on scallop larvae producing high mortality as well as the clinical signs typical of vibriosis. Therefore, the use of bacterial cell staining with 5-DTAF to study the chronology of invasive processes caused by Vibrio species is a time-saving protocol, highly recommended to follow the vibriosis on mollusc larvae.The uropaeus , and V. V. bivalvicida VPAP30 strain mediated by the production of ECPs was evaluated. Our results demonstrated that cell-free ECPs of the VPAP30 strain are involved in the pathogenic action on scallop larvae, causing a mortality of 40% of challenged larvae after 48 h of exposure. The results of this study demonstrate that the extracellular toxigenic activity exhibited by this strain is mainly mediated by the production of heat stable compounds, causing larval necrosis and the detachment of ciliary cells, consistent with previous reports, which demonstrated that Vibrio strains can produce heat stable ciliostatic toxins and proteinases that degrade larval tissue  produces some extracellular compounds, including a cytolysin/haemolysin (V. europaeus strain (formerly V. tubiashii ATCC 19105) on oyster larvae, detecting the production of a metalloprotease encoded by the vtpA gene. It must be noted that a metalloprotease encoding gene similar to vtpA was detected in the genome of this pathogenic strain (formerly V. tubiashii VPAP30)  . Moreoveilyticus , but pre strains .V. bivalvicida strain outside Europe, being recovered from a massive mortality episode in a commercial larval culture of the scallop A. purpuratus in Chile. Our results demonstrated that V. bivalvicida VPAP30 is highly pathogenic to scallop larvae exhibiting an invasive activity as well as a production of toxigenic heat-stable ECPs and producing the typical clinical signs of vibriosis. In addition, this strain produced on experimentally infected scallop larval cultures very similar symptoms to those observed during the massive mortality event in the Chilean commercial hatchery strongly suggesting that this strain was the causative agent of the mass mortalities occurring in the referred event. Then, we have demonstrated for the first time the occurrence of a highly pathogenic strain of V. bivalvicida in a commercial hatchery of scallop A. purpuratus, suggesting this species can become in a pathogen of major concern for the Chilean pectinid industry. The proper identification of this bacterial pathogen causing epizootics in larval cultures is essential for developing efficient epidemiological management strategies to prevent and control outbreaks in Chilean intensive scallop larvae husbandry.In conclusion, this is the first isolation of a RR designed the study, isolated the bacterial strain, performed all challenge assays, drafted the manuscript, and is the corresponding author and the primary contact. CM contributed significantly to the design, drafting, revisions, and interpretation of data. JR designed the study together with RR, supervised the study and advised molecular analysis and interpretation. JD and JB helped to analyze the MLSA analysis and genomic comparisons and in silico DNA-DNA hybridizations. All authors have made intellectual contributions to the work and approved it for publication.The authors declare that Invertec Ostimar company only contributed to permitting the scallop larvae collection, and had no role in the study.


Nitrogen (N) losses from cattle feedlots are of concern due to loss of valuable N and enrichment of the atmospheric N pool. Nutritional methods to decrease such losses would have economic and environmental benefits. One method to decrease N losses is by increasing carbon (C) on the pen surface. The most cost effective method of decreasing N losses with C may be feeding diets lower in digestibility compared to adding C directly to pens. Therefore, three experiments evaluated feeding corn bran (which is less digestible than corn) as either 0, 15, or 30% of the diet. The 15- and 30%-bran diets increase organic matter (OM) excretion by approximately 0.5 and 1.0 kg per steer per day, respectively. Compared with no bran, feeding 15 and 30% decreased feed efficiency by 7.8 and 10.4%, respectively. Nutrient balance was assessed in two trials from October through May and in one trial from June to September. During the trials from October to May, N losses were decreased by 14.5 and 20.7% for the 15- and 30%-bran diets compared with no bran. Feeding 15 or 30% bran did not influence N losses in the experiment from June to September. Increasing the C:N ratio of manure prior to cleaning open-dirt feedlots had variable results depending on time of year.


Hearing impairment is one of most frequent birth defects, which affects nearly 1 in every 1,000 live births. However, the molecular etiology of non‐syndromic deafness in China is not well studied. Here, we have investigated the presence of mutations in three genes commonly mutated in non‐syndromic deafness patients in Shanxi Province, which has the highest frequency of birth defects in China.GJB2, SLC26A4 (PDS), and mtDNA 12S rRNA) of all individuals enrolled were analyzed by Sanger sequencing.In total, 1,201 unrelated non‐syndromic deafness patients and 300 healthy individuals were enrolled. The hearing ability was confirmed by audiologic evaluation. Three major deafness‐related genes  in the patient group, with c.235delC, a hotspot mutation, accounting for 10.99% . Moreover, 11 new GJB2 mutations were identified. SLC26A4 mutations accounted for 9.33%  in the patient group, with IVS7‐2A>G as the most prevalent mutation accounting for 4.75% . In addition, 15 patients (1.25%) were found to carry mtDNA 12S rRNA c.1555A>G mutation, while only two cases had the mtDNA 12S rRNA c.1494C>T.The results showed that GJB2 and c.919‐2A>G (IVS7‐2A>G) in SLC26A4 were the highest frequency pathogenic variants in Shanxi Province. Taken together, our data will enrich the database of deafness mutations and will help clinical diagnosis, treatment, and genetic counseling of hearing impairment.In our research, it was found that c.235delC in  More than half of childhood hearing impairment is caused by genetic defects. Among these, 30% of cases are syndromic with other abnormalities, while approximately 70% are non‐syndromic, in which the hearing impairment is the only distinctive clinical feature in these patients . Exhilaratingly, combining the GWAS analysis and the next‐generation sequencing technique, more than 150 loci have been discovered to date, including 54 autosomal dominant loci, 71 autosomal recessive loci, 5 X‐linked loci, two modifiers, and 1 Y‐linked locus  account for 50% of autosomal recessive hearing loss (HL) in most populations in the world  are the second most frequent cause of non‐syndromic hearing loss. Mutations in SLC26A4 are normally associated with large vestibular aqueduct syndrome, showing congenital temporal bone malformation of the vestibular aqueduct as well as Mondini dysplasia  are associated with maternally inherited sensorineural hearing loss. The mtDNA of human is double‐stranded, encoding 13 protein subunits, two ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs)  are the major cause of the genetic deafness in China  in 1,201 patients with non‐syndromic deafness in Shanxi Province, which has a high incidence of HL. Our data suggested that 21.23%, 9.33%, and 1.25% of the patients carry mutations in GJB2, SLC26A4, and MT‐RNR1 in Shanxi Province. These data will enrich the database of deafness mutations and provide the standard for clinical diagnosis, treatment, and genetic counseling in Shanxi. Also, it will contribute to development of a diagnosis kit for patients with non‐syndromic hearing loss in Shanxi Province.Early diagnosis, intervention, and treatment are critical for controlling the genetic hearing loss. Although many studies indicated that, similar to Western countries, mutations in 22.1This study was approved by the ethics committee of the Second Hospital of Shanxi Medical University.2.2In total, 1,201 non‐syndromic deafness subjects from Shanxi Province, who were enrolled from the School for deaf‐mute and special school, were recruited. Family history was obtained by consulting subjects or their parents. Individuals of the control group without family history were enrolled from the Blood Transfusion Department of the Second Hospital of Shanxi Medical University. In the patient group, there were 698 males and 503 females, with an average age of 19.25 years, ranging from 0.5 to 38 years. Both patients and control individuals were Han Chinese and have signed informed consent by themselves or their parents according to the protocol approved by the Ethics Committee of the Second Hospital of Shanxi Medical University.2.3All patients or their guardians completed the survey by answering questions and filling in the "deafness questionnaire survey form." The details were as follows: (a) basic information: name, gender, date of birth, nationality, marital status, contact information, etc.; (b) morbidity status: age of onset, presence of other associated symptoms , related medical history , history of use of aminoglycoside antibiotics, long‐term noise exposure, otitis media or traumatic history of the ear, etc.; (c) the development condition of language, whether wearing hearing aid or cochlea, etc.; and (d) whether merging other systemic diseases , family history, and eclampsia pregnancy history: the infecting status of mothers during pregnancy  or the presence or absence of dystocia.2.4Ear examination and audiological evaluations including pure‐tone audiometry, immittance testing, auditory brainstem response, and auditory steady‐state response were performed for all hearing impairment patients. Physical and neurological examination was carried out with special attention to renal and ophthalmologic differences to exclude those with syndromic deafness. Only non‐syndromic deafness subjects were kept in the study.2.5GJB2, SLC26A4, and mtDNA rRNA) were investigated in all the subjects. Genomic DNA was extracted from the peripheral blood using QIAGEN Blood DNA Kits  following the manufacturer's instructions. Gene‐specific primers for amplifying exons of GJB2 (NM_004004.5) and parts of SLC26A4 (NM_000441.1) were designed by Primer 5.0 software, and the sequences are listed in Table SLC26A4 (NM_000441.1) were sequenced using the conditions reported by Sagong, Baek, Baek, Lee, and Kim  for diagnosis of enlarged vestibular aqueduct (EVA) or inner ear malformation. The diagnosis standard is diameter >1.5 mm at the midpoint between the common crus and external aperture  were found with hearing loss under the age of 3 years (they were prespeech deafness). Forty‐seven cases of deafness (3.91%) were found after 3 years old (they were postspeech deafness). Thirty‐four patients were found with hearing loss after high fever, accounting for 2.83%. Twenty‐three patients provided a history of the usage of aminoglycoside drugs. Sixty‐three patients were infected with CMV, and eight cases had HL, accounting for 12.7% (8/63). Seventy‐one patients had the history of injury, resulting in HL. There were 27 cases of deafness with unclear medical history. In the 1,201 patients with non‐syndromic deafness, 1,146 were full‐frequency hearing loss, 38 were high‐frequency hearing loss, and 17 were low‐frequency hearing loss. There were 786 cases of binaural hearing impairment and 415 cases of monaural hearing impairment. Immittance testing revealed that all patients evoked otoacoustic emissions.GJB2 mutations were found in 21.23%  of the patients; SLC26A4 mutations were found in 9.33%  of the patients; and 12S rRNA mutations were found in 1.42%  of the patients. In 17 mtDNA 12S rRNA mutation subjects, 15 carried the m.1555A>G and two carried the m.1494C>T  carried a single mutant allele and 4.33%  carried two mutant alleles. Four patients showed compound heterozygous mutations carrying c.35delG;c.235delC, c.176‐191del16; c.299‐300delAT, c.35delG;c.176‐191del16, and c.235delC;c.299‐300delAT, respectively.In patients with Among the 300 healthy individuals, only two subjects were found to carry heterozygous pathogenic mutation c.235delC with a frequency of 0.67% (2/300); two other variants, c.79G>A and c.341A>G, accounted for 28.67% (86/300) and 14.00% (42/300), respectively. c.79G>A and c.341A>G were polymorphisms that could lead to nonsynonymous changes: p.V27I and p.E114G, respectively  of the patients had two mutant alleles (homozygote or compound heterozygote).Mutations in s Figure . c.919‐23.3mt12S rRNA gene mutation was reported as one of major causes of non‐syndromic deafness, the mutation of this gene was investigated . It was reported that GJB2 mutations are also associated with a large proportion of non‐syndromic deafness patients in Austria and Africa . However, this mutation was not found in the 300 healthy individuals. A previous study has shown that c.109G>A was a polymorphism  cases in the patient group and three individuals in the control group were found to carry mutations of mtDNA 12S rRNA mutations found in the present study was lower than either that reported by Guo et al. . This suggests that CMV infection is an important factor in the occurrence of deafness, but its mechanism has not been fully elucidated at present. Most scholars believe that it mainly causes deafness by the development of inhibiting sensory nerve.5GJB2 and c.919‐2A>G in SLC26A4 were the most prevalent pathogenic variants in Shanxi Province. This result is consistent with previous reports. The cause of deafness is very complex. Deafness is influenced by genetic and environmental factors. It is an arduous task to identify the pathogenic mechanism. However, more and more genes related to deafness were found. Epidemiological studies revealed that the mutation frequency of the three common deafness genes varies in different regions. The results of our research will lay a foundation for the prevention of deafness in Shanxi Province. They will enrich the database of deafness mutations and make a contribution to clinical diagnosis, treatment, and genetic counseling in non‐syndromic hearing loss in future.In our research, it was found that c.235delC in None declared.


We evaluated if preoperative TG levels affected postoperative levels and if other factors may influence the optimal time to check postoperative TG.  This is a prospective, observational pilot study. We approved and enrolled 50 subjects ≥ 19 years scheduled for total thyroidectomy and measured serum TG, thyroglobulin antibody (TG ab), and TSH preoperatively and post thyroidectomy at 7-14 days, 4 and 6 weeks, and 3 months in subjects with benign pathology, with additional 6- and 12-month measurements in subjects with thyroid cancer. p = 0.0006) group. In the benign group, 76.5% (13/17) of subjects had an undetectable TG < 0.2 ng/mL by 12 weeks postoperatively. In the malignant group, 70.6% (12/17) of those who did not receive RAI therapy and 25% (1/4) of those who did receive RAI had undetectable TG < 0.2 ng/mL by 12 weeks. Subset analysis showed 94.1% (16/17) of the benign, 70.6% of the malignant without RAI, and 50% (2/4) of the malignant with RAI achieved TG < 1.0 ng/mL by 6 weeks postoperatively. Four subjects in the malignant group reached undetectable TG levels as early as 7-14 days postoperatively.  Preoperative TG was significantly higher in the benign (median 167.5 ng/mL vs 30.8 ng/mL) than in the malignant (NCT02347683. Preoperative TG levels did not predict the risk of malignancy nor time to TG nadir postoperatively. We did not find a difference in TG elimination half-life between the benign and malignant groups. The median time to reach undetectable TG levels in both benign and malignant groups who did not receive RAI therapy was 12 weeks. However, those with preexisting hypothyroidism and hyperthyroidism had lower levels of TG overall in the malignant group which can be taken into consideration besides other known factors that can affect TG levels post thyroidectomy. This trial is registered with Clinicaltrials.gov  Thyroglobulin (TG) is a dimeric glycoprotein 660 kDa) synthesized and stored in the follicular cells of normal thyroid tissue and regulated by Thyroid-Stimulating Hormone (TSH) . Serum T60 kDa syAdditionally, TG is used in assessing response to therapy for well-differentiated thyroid cancer. A TG of <0.2 ng/mL is consistent with an excellent response to therapy and predicts risk of recurrence of 1-4%. A TG of 0.2-1.0 ng/mL is considered an indeterminate response to therapy and has a predicted risk of recurrence of 15-20%, while a biochemically incomplete response to therapy is a suppressed TG > 1 ng/mL or a stimulated TG > 10 ng/mL with predicted risk of recurrence of 20% [TG, however, is not routinely tested preoperatively, and guidelines do not recommend checking TG as part of the preoperative workup . Many beTG is clearly an important part of the evaluation, treatment, and long-term follow-up of DTC; however, optimal timing to check the TG and additional factors that may affect it have not been extensively studied. In this study, we evaluated whether preoperative TG predicted postoperative TG. Additionally, we evaluated other factors that may influence the optimal time to check postoperative TG.This is a prospective observational pilot study. Local IRB approval was obtained. We recruited 50 consecutive adult (age ≥ 19 years) subjects who were planning to undergo total thyroidectomy from March 2015 through February 2016. Subjects consented preoperatively and baseline TG, TG antibody (TG ab), and TSH were obtained prior to surgery. Postoperatively, they were divided into two groups  based on final pathology. All patients underwent total thyroidectomy with the same group of head and neck surgeons. Subjects were excluded if they were pregnant or lactating. Those with benign pathology were considered the nonmalignant control. They were compared to those with malignant pathology to determine if there was a difference in clearance of TG in the benign and malignant groups. Postoperatively, blood was obtained at predetermined intervals for both the benign and malignant groups. The benign group had TG, TG ab, and TSH at 7-14 days, 4 weeks, 6 weeks, and 3 months postoperatively. The malignant group had TG, TG ab, and TSH at 7-14 days, 4 weeks, 6 weeks, 3 months, 6 months, and 12 months postoperatively. If they had positive TG ab which persisted postoperatively, subjects were excluded since TG ab renders the TG uninterpretable. At our institution, it is standard of care to follow up subjects with thyroid cancer with postoperative ultrasound between 3 and 6 months to monitor any residual or recurrence.All serum TG and TG Abs were assessed using the Siemens Immulite 1 and were performed at the University of Nebraska Medical Center. The Siemens Immulite 1 is a solid-phase enzyme-labelled, chemiluminescent sequential immunometric assay with a functional sensitivity for TG < 0.2 ng/mL and TG ab < 20 IU/mL. Ultrasensitive TSH assay was done using Beckman Coulter DxI 800 with a reference range of 0.4-5.0 mcIU/mL.p < 0.05 significance level and correspond to a hazard ratio of 0.6986. These results are based on the assumption that the hazard rates are proportional. For our study, we enrolled 50 subjects since it is a pilot study.PC SAS version 9.4 was used for all analyses. A time to event analysis using the LIFETEST procedure in SAS was used where the nadir is defined as the event of interest. Time to nadir was compared for subjects with benign pathology versus those with thyroid cancer. Other variables that were compared for those in the benign group versus those in the cancer group include preoperative TG, stage, age (<45 versus ≥45 years), serum creatinine, gender, race, RAI treatment, and histologic type. Chi-square and Fisher's exact tests were used for the categorical variables, and the nonparametric Wilcoxon test was used to compare the continuous variables. Descriptive statistics are provided for all variables. Scatterplots of TSH and TG and bar charts representing the percentage of subjects who reached TG nadir at different time points are provided. We calculated the sample size using a one-sided log-rank test which showed that if 294 subjects are enrolled, that will achieve 80% power at Fifty subjects who were planning to undergo total thyroidectomy signed consent. Complete data analysis was performed on 45/50 subjects enrolled . Two subp = 0.82). Median age was 52.9 years in the benign and 50.7 years in the malignant group (p = 0.65). Sixty percent (15/25) of the malignant histologic diagnoses was classic papillary, 28% (7/25) was follicular variant papillary thyroid cancer (FVPTC), and 12% (3/25) was encapsulated variant FVPTC (NIFTP). Since the study occurred prior to NIFTP classification, NIFTP was included in the malignant group. The majority, 64% (16/25), were categorized as AJCC 7th Edition, stage 1, 16% (4/25) stage 2, 16% (4/25) stage 3, and 4% (1/25) stage 4. When evaluating ATA risk stratification, 64% (16/25) had a low risk, 32% (8/25) had an intermediate risk, and 4% (1/25) had a high risk of recurrence. RAI therapy was administered to 31.8% (7/25) of the subjects. At the conclusion of the study, 68% (17/25) of subjects had an excellent response to therapy, 28% (7/25) had an indeterminate response, and 4% (1/25) had a biochemically incomplete response. None of the subjects had structurally incomplete response to therapy (Twenty subjects had benign pathology and 25 subjects had thyroid cancer. Seventy-five percent (15/20) of the benign and 72% (18/25) of the malignant group were female ( therapy .p = 0.0006)  preoperative TG was 167.5 ng/mL (30.2-2349 ng/mL) in the benign group and 30.8 ng/mL (0.4-264 ng/mL) in the cancer group ( 0.0006) .Of the 45 total subjects, six had persistent positive TG ab postoperatively, so they were excluded from the final analysis. One additional subject had preoperative lab and surgery data available but was unable to return for follow-up after surgery. Overall, 38 subjects were evaluated in the final postoperative TG analysis .In the malignant group, seven subjects received RAI therapy 6-12 weeks postoperatively. These subjects were evaluated as a subgroup of the malignant group. Overall, TG prior to RAI therapy ranged from 0.2 to 2.0 ng/mL. Three subjects had TG ab postoperatively, making the TG uninterpretable, so they were excluded. All the subjects who underwent RAI therapy received only one dose of I-131 ranging from 100 to 180 mCi.Serum TSH was measured at each time point with TG and TG ab. The relationship of TSH and TG is shown in p = 0.90)  time required to achieve undetectable TG levels in both benign and malignant groups who did not receive RAI therapy was 12 weeks ; malignant: 84 days ). In subjects who received RAI therapy, median time to achieve undetectable TG was 182.5 days  ( = 0.90) .p = 0.84)  time to achieve TG < 1.0 ng/mL was 6 weeks for the benign group: 42 days , and 4 weeks for the malignant group who did not receive RAI therapy: 28 days , while it was 8 weeks for the malignant group who received RAI therapy: 56 days  ( = 0.84) .Time to achieve TG nadir for each subject in both the groups is shown in Determining the optimal timing of postoperative serum TG measurement is important since it plays an important role in clinical decision making. Guidelines recommend a broad testing window of 3-12 weeks postoperatively; this recommendation is largely based on previous studies of TG elimination half-life. Previous studies evaluating the elimination half-life of TG vary from 6-96 hours and the variation was thought to be due to differences in methodology to measure TG levels. One study reported TG is eliminated through the liver and its half-life following total thyroidectomy was 65.2 hours (range: 36.9-86.6 hours) when measured by RIA to eliminate interassay variations. The TG level was noted to decrease to less than 5-10 ng/mL 25 days after thyroidectomy, or after 7 to 10 half-lives in 11 patient samples [In our study, the median time to achieve an undetectable TG level postoperatively in both the benign and malignant groups who did not receive RAI therapy was 12 weeks. However, not surprisingly for those who received RAI therapy, it can take as long as 6 months for undetectable TG levels. If using a TG cutoff of <1.0 ng/mL, median time to goal is 4-8 weeks. This is consistent with the range found in previously published studies –12. It sBy obtaining preoperative TG levels, this allowed us to evaluate the role of preoperative TG levels in predicting the risk of malignancy. We found no correlation between preoperative TG levels and malignancy. In fact, two subjects in the benign group had markedly elevated TG values > 2000 ng/mL. Both of these patients had no evidence of preexisting thyroid disease, had negative TG ab, and had homogenous appearing thyroid parenchyma separate from the bilateral thyroid nodules with indeterminate thyroid FNA biopsies (Bethesda III and IV). The reason for the marked elevation in TG is unclear. This observation supports the ATA recommendation #3 against preoperative TG testing to predict risk of malignancy .Conversely, we had two subjects with thyroid cancer who had preoperative TG levels < 0.2 ng/mL. Both subjects had preexisting hypothyroidism and were on levothyroxine therapy. These two subjects were two of the four subjects that had undetectable TG at 7-14 days postoperatively. Of the two remaining subjects with undetectable TG levels in the malignant group at 7-14 days postoperatively, one had preexisting hyperthyroidism due to toxic multinodular goiter and was on methimazole preoperatively. The potential mechanism for these findings could be that subjects with hypothyroidism on chronic levothyroxine therapy can have small gland volume and low endogenous thyroid hormone and TG production at baseline leading to overall low endogenous TG levels that are eliminated quickly after surgery. Similarly, for subjects with hyperthyroidism, antithyroid therapy also suppresses endogenous TG production potentially allowing for lower baseline levels and more rapid elimination of endogenous TG. These possible mechanisms need to be studied further.This is an observational pilot study, limited by small numbers; however, it looked at both pre- and postoperative TG levels in subjects with both benign and malignant pathologies. We did not find a difference in TG elimination half-life between the benign and malignant groups. We also did not show correlation between preoperative TG and malignancy. However, those with preexisting hypothyroid and hyperthyroidism had lower levels of TG overall. The median time to reach undetectable TG levels in both the benign and malignant groups who did not receive RAI therapy was 12 weeks if using TG < 0.2 ng/mL as a functional sensitivity cutoff.


Vigna mungo var. mungo) is an important pulse crop in Asia. The cowpea weevil  is a stored-seed insect pest (seed weevil/bruchid) that causes serious postharvest losses in pulse crops, including black gram. In this study, we constructed a high-density linkage map for black gram and identified quantitative trait loci (QTLs) for C. maculatus resistance. A recombinant inbred line (RIL) population of 150 lines from a cross between BC48  and TC2210  were used to construct a linkage map of 3,675 SNP markers from specific-locus amplified fragment sequencing. The map comprised 11 linkage groups spanning 1,588.7 cM with an average distance between adjacent markers of 0.57 cM. Seeds of the RIL population grown in 2016 and 2017 were evaluated for C. maculatus resistance through two traits; the percentage of damaged seeds (PDS) and infestation severity progress (AUDPS). Inclusive composite interval mapping identified three QTLs each for PDS and AUDPS. Two QTLs, qVmunBr6.1 and qVmunBr6.2, mapped about 10 cM apart on linkage group 6 were common between PDS and AUDPS. Comparative genome analysis revealed that qVmunBr6.1 and qVmunBr6.2 are new loci for C. maculatus resistance in Vigna species and that genes encoding a lectin receptor kinase and chitinase are candidates for qVmunBr6.2. The high-density linkage map constructed and QTLs for bruchid resistance identified in this study will be useful for molecular breeding of black gram.Black gram ( Vigna mungo (L.) Hepper) is an important legume crop of Asia. It is cultivated as a component of various cropping systems that cover over four million hectares, principally in India, Myanmar, Pakistan, Bangladesh and Thailand. In South Asia, dry black gram seeds are mainly consumed as a thick soup, while seed flour is used to prepare several dishes. In Thailand and Japan, black gram seeds are popular in the sprout industry. Nonetheless, the average seed yield of blackgram is very low, e.g. about 650–800 kg/ha in Thailand and India. In addition, after harvest, black gram seeds are frequently destroyed by bruchids (seed weevils) (Coleoptera: Bruchidae). Generally, the seeds of all Vigna crops are attacked by bruchids. The two most important and damaging bruchid species for Vigna crops are the cowpea weevil  and azuki bean weevil 1. These two bruchid species are widely distributed across nearly all continents due to international seed trading. Bruchid infestation of legume crops initially occurs in the field where female bruchids lay eggs on young pods and larvae bore through the pods into the seeds, in which they grow and develop into adults by consuming seed nutrients. The infestation starts in the field and continues during storage. After harvest, the adult bruchids emerge from the seeds and start a new infestation by laying eggs directly on seeds, which can result in the total loss of a seed lot within 2–4 months1. Chemical fumigation is mostly used to control the bruchids, but it is not practical for small-scale farmers and traders and is unsafe for human health and the environment. The most practical and sustainable method for managing bruchid infestation is the use of resistant cultivars.Black gram  is completely resistant to C. chinensis, but highly susceptible to C. maculatus7. In contrast, its wild progenitor, Vigna mungo var. silvestris Lukoki, Maréchal & Otoul, is resistant to both of these bruchid species7. Compared with cultivated black gram, wild black gram shows a lower percentage of adult emergence (PAE), lower percentage of damaged seeds (PDS), and longer developmental period (DP) for C. maculatus8. Its resistance is due to antibiosis of seeds9. Based on PAE evaluation, it was reported that the resistance to C. maculatus in V. mungo var. silvestris is controlled by two dominant duplicate genes, designated Cmr1 and Cmr26. Quantitative trait loci (QTLs) for the resistance were located on a linkage map of a recombinant inbred line (RIL) population (V. mungo var. silvestris × V. mungo var. mungo) using 428 markers (381 dominant and 47 co-dominant markers)9. Two QTLs for PAE, Cmrae1.1 and Cmrae1.2, were identified on linkage groups (LGs) 3 and 4, respectively, while six QTLs were identified for DP; two (Cmrdp1.1 and Cmrdp1.2) on LG1, three  on LG2, and one (Cmrdp1.1) on LG109. Nonetheless, none of these QTLs have been used in genetic improvement of the traits because of a lack of closely linked markers.Sources of bruchid resistance have been reported for some 10. Thus, in marker-assisted selection, high-resolution mapping of a trait should be conducted to identify markers closely linked to the target trait. High-resolution mapping is also useful to identify the gene or candidate gene underlying the trait. Currently, advanced sequencing technologies enable scientists to directly detect thousands of single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) in organisms rapidly at low cost. Several sequencing-based genotyping methods have been developed such as genotyping-by-sequencing (GBS)11, restriction-associated DNA sequencing (RAD-seq)12 , diversity array technology sequencing (DArTseq)13 and specific-locus amplified fragment sequencing (SLAF-seq)14.Molecular breeding by marker-assisted selection relies on DNA markers closely linked to the trait of interestC. maculatus.In this paper we report the development of a high-density linkage map for black gram using the SLAF-seq technique and the identification of QTLs controlling seed resistance to 2 individual ranged from 1,178,049 to 2,363,336 with an average of 2,491,876. After read clustering, a total of 577,828 SLAFs were detected, and the average sequencing depth was 35.65-fold, 26.00-fold and 9.28-fold for BC48, TC2210 and the progeny, respectively.After preprocessing, 79.49 Gb of raw data containing 399.60 M reads were generated. On average, the Q30  was 93.02% and the GC content was 39.76%. The numbers of reads for TC2210 and BC48 were 12,553,056 and 13,266,999, respectively. The read numbers for each FFrom the 577,828 SLAFs, after filtering SLAF markers lacking parent information and showing low read depth, 30,978 polymorphic markers were successfully genotyped and grouped into eight segregation patterns viz., ab × cd, ef × eg, hk × hk, lm × ll, nn × np, aa × bb, ab × cc, and cc × ab. In total, there were 8,913 markers showing the segregation pattern aa × bb.After careful screening, 4,588 markers were used for genetic linkage analysis, among which 3,675 80.1%) were clustered into a linkage map of 11 LGs  was conducted in the RIL population to locate QTLs for qCm_PDS6.1) and two QTLs for AUDPS (qCm_AUDPS6.1 and qCm_AUDPS6.2). All of these QTLs were on LG6. qCm_PDS6.1 and qCm_AUDPS6.1 were located very near to each other (about 1 cM apart) on LG6 and each explained about 28% of the total variation of each trait in the RIL population. qCm_AUDPS6.2 was about 12 cM away from qCm_PDS6.1 and qCm_AUDPS6.1. It accounted for 17.37% of the total variation of AUDPS in the RIL population.In 2016, ICIM identified one QTL for PDS (qCm_PDS6.1 qCm_AUDPS6.1 and qCm_AUDPS6.2) were also found in 2016, while the other three (qCm_PDS2.1 and qCm_PDS6.2 for PDS and qCm_AUDPS7.1 for AUDPS) were found only in 2017. qCm_PDS6.1, qCm_AUDPS6.1 and qCm_AUDPS6.2, which were detected in 2017, showed similar percentages of variance explained (PVEs) to those in 2016. qCm_PDS2.1, qCm_PDS6.2, and qCm_AUDPS7.1 were on LGs 2, 6 and 7, respectively. qCm_PDS2.1 and qCm_AUDPS7.1 had PVEs of 7.95% and 7.28%, respectively, while qCm_PDS6.2 had a PVE of about 15%.In 2017, six QTLs for resistance were detected; three for PDS and three for AUDPS. Of the six QTLs, three (qCm_PDS6.1 and qCm_PDS6.2 and two for AUDPS (qCm_AUDPS6.1 and qCm_AUDPS6.2). These QTLs were mapped to the same or very similar positions to those in the years 2016 and/or 2017. They accounted for 22.40%, 17.36%, 30.04%, and 19.60% of the trait variation in the combined data.In the case of the combined data, four QTLs were detected for resistance; two for PDS 16. The high-density linkage map developed here comprised 3,675 SNP-based SLAF-seq markers that resolve the 11 haploid chromosomes of black gram C. maculatus resistance in black gram in this study was only about 40%. This indicated that resistance is principally affected by the environment. This heritability value in this study is very low compared with the value previously estimated for C. maculatus resistance by9, who reported up to 99% heritability for resistance in black gram. The contrasting results between our study and that of 9 may stem from (i) the difference in the sources of resistance and/or (ii) differences in biotypes (strains) of C. maculatus. Additional study is necessary to explain the contrasting results.The narrow-sense heritability estimated for C. maculatus resistance in the wild black gram TC2210  and six for developmental period. The QTLs Cmrae1.1 and Cmrae1.2 were mapped to different LGs. As a result, these QTLs appear to be different from the QTLs qVmunBr6.1 and qVmunBr6.2 for C. maculatus resistance in TC2210, which were mapped to the same LG 21. We compared the major QTLs for C. maculatus identified in these studies and our study (qVmunBr6.1 and qVmunBr6.2) by conducting BLASTN analysis of the SSR marker sequences linked to the QTLs against the reference genomes of mungbean22 and azuki bean23. We found that the chromosome locations of qVmunBr6.1 and qVmunBr6.2 were chromosome 8 of mungbean and chromosome 8 of azuki bean, and were different from the chromosome locations of the bruchid resistance QTLs in other Vigna species , a 173.7 kb region of chromosome 8 of azuki bean  and a 150.9 kb region of cowpea chromosome 7  (Supplementary Table LOC106770636 and Vigun07g218900 encoding lectin receptor kinase (LecRK) were present in the 160.2 kb and 150.9 kb regions of mungbean and cowpea, respectively. The seed lectins of some legume species have been shown to be highly toxic to C. maculatus27. Because LecRK possesses a lectin domain, it may be toxic to C. maculatus. In addition, we found that the genes LOC106771180 (Chr08: 40114473..40115564), Vigan.08G086800.01  and Vigun07g219300  encoding chitinase were located very close to the 160.2 kb region of mungbean, 173.7 kb region of azuki bean and 150.9 kb region of cowpea, respectively. A chitinase isolated from cowpea seeds was shown to affect the development of C. maculatus larvae, although it did not much affect their survival28. Chitinase is among the seed chemicals that are associated with resistance to C. chinensis in mungbean29. Recently, soybean seed coat chitinase was shown to be highly toxic to C. maculatus30. Thus, the genes for LecRK and chitinase can be considered candidate genes at the QTL qVmunBr6.2 for C. maculatus resistance in black gram.Because a reference genome sequence of black gram was not available and there is high genome conservation between species in the genus C. maculatus, while TC2210 is a wild blackgram from India and is resistant to C. maculatus7. TC2210 showed a lower percentage of damaged seeds and slower progress of seed damage caused by bruchids compared with BC48.A RIL population of 150 lines developed by a single-seed descent method from the cross ‘BC48’ × ‘TC2210’ was used in this study. BC48 is a cultivated blackgram from Thailand and is susceptible to 9 RILs (one plant per line) and their parents were grown under field conditions using 0.5 × 0.5 m spacing at Kasetsart University, Kamphaeng Sean Campus (KU-KPS), Nakhon Pathom, Thailand during October to December. At maturity, the pods of each plants were harvested separately for bruchid evaluation. Genomic DNA was extracted from young leaves of the F9 RILs and parents using a CTAB method31.In 2016, the F10 RILs and the parents were grown in a randomized complete block design with two replicates under field conditions at the KU-KPS during June to August. The spacing between rows was 0.5 m and the spacing between plants in the same row was 0.25 m. In each replicate, each entry comprised 10 plants, five of which were randomly selected and harvested. The seeds harvested from each individual plant were used for bruchid resistance evaluation.In 2017, the F14 with minor modifications. Briefly, a pilot experiment for SLAF analysis was performed using the reference genome of mungbean22. Based on the results of the pilot experiment, a SLAF library was prepared. Genomic DNA from each entry was digested at 37 °C with RsaI and HaeIII , incubated with the Klenow fragment (3′ → 5′exonuclease) (NEB) and dATP at 37 °C to add a single-nucleotide A overhang to the digested fragments, and the A-tailed DNA fragments were then ligated to Duplex Tag-labelled sequencing adapters  using T4 DNA ligase. PCR was performed using diluted restriction-ligation DNA samples, dNTPs, Q5® High-Fidelity DNA Polymerase and PCR primers  . The PCR products were then purified using Agencourt AMPure XP beads  and pooled. The pooled samples were separated by 2% agarose gel electrophoresis. Fragments ranging from 314 to 414 bp (with indexes and adaptors) in size were excised and purified using a QIAquick gel extraction kit . The gel-purified products were diluted. Pair-end sequencing (each end 125 bp) was performed using an Illumina HiSeq 2500 system  according to the manufacturer’s recommendations at the Biomarker Technologies Corporation .Genomic DNA extracted from the parents and 150 RILs was used for SLAF library construction and sequencing as per22 using BWA. SLAF marker identification and genotyping were performed as per14. Low-quality reads  were eliminated and the raw reads were assigned to 150 F2 individual samples according to the duplex barcode sequences. After trimming each high-quality read, the clean reads were clustered together according to their sequence identities. Sequences mapping to the same locus with over 90% identity were defined as one SLAF locus. SNP loci between the two parents were detected and SLAFs with >3 SNPs were removed. The alleles of each SLAF were defined according to the parental reads with a sequence depth >6-fold and offspring reads with a sequence depth >2-fold. In black gram, which is a diploid species, one locus contains a maximum of four genotypes; thus, SLAF loci with >4 alleles were discarded and SLAFs with 2, or 3, or 4 alleles were considered potential markers. Polymorphic markers were grouped into eight segregation patterns14, but only markers showing the segregation pattern aa × bb were selected. To ensure the genotyping quality, genotype scoring was conducted using a Bayesian approach as described by14. Then, three processes were performed to screen for high-quality markers; (i) markers with average sequence depths <6-fold in the parents and <1-fold in the progeny were discarded, (ii) markers with >40% missing data were removed, and (ii) markers with significant segregation distortion (P < 0.01) were initially excluded from the genetic map construction and then added later as accessory markers.The reads generated from sequencing were compared with the mungbean reference genome sequence32 was utilized to order the SLAF markers and correct genotyping errors within LGs. Firstly, recombinant frequencies and LOD scores were calculated by two-point analysis to infer linkage phases. Then, enhanced Gibbs sampling, spatial sampling and simulated annealing algorithms were combined to conduct an iterative process of marker ordering34. The error correction strategy of SMOOTH35 was implemented according to the parental contribution of genotypes, and a k-nearest neighbour algorithm was applied to impute missing genotypes36. Skewed markers were added into this map by applying a multi-point maximum likelihood method. Map distances were calculated using Kosambi’s mapping function37.Polymorphic SLAF markers were partitioned primarily into LGs based on their locations on the mungbean reference genome. Next, modified logarithm of odds (MLOD) scores between markers were calculated to further confirm the robustness of the markers for each LG. Markers with MLOD scores <6 were filtered prior to ordering. The HighMap strategyC. maculatus was reared on seeds of the susceptible mungbean cultivar ‘Kamphaeng Saen 1’ and kept in a room at 30 °C and 50% relative humidity. Evaluation of C. maculatus resistance was conducted as per38 with minor modifications. The seeds harvested from each plant were subjected to resistance evaluation. Forty seeds from each plant were put in a plastic box. Then, 10 pairs  of newly emerged C. maculatus adults were introduced into the box for egg laying for 7 days and then removed. The infested seeds were maintained at 30 °C and 50% relative humidity. The numbers of seeds damaged by the bruchids were recorded at 40 days after bruchid introduction (DAI), and then at every 5 days until 80 DAI. Each time the seeds were assessed, the damaged seeds were removed from the boxes. Seeds of both parents were also included in the resistance evaluation. The cumulative number of total seeds damaged by the bruchid (seeds with holes) at each counting date was calculated and converted into a percentage. Then, the percentage of damaged seeds was used to calculate the area under disease progress stair (AUDPS)39. AUDPS is an improved version of the area under disease progress curve (AUDPC), which indicates progression of disease severity in plants. In this study, AUDPS was used as an indicator for the progression of bruchid infestation severity. The percentage of damaged seeds at 80 DAI and the AUDPS value of each RIL were used for data analysis.A culture of h2) of PDS and AUDPS was determined for the RIL population grown in the replicated experiment (F10). Analysis of variance of PDS and AUDPS was conducted, and then the h2 of each trait was calculated using the following formula:r is the number of replicates.The narrow-sense heritability (40 implemented in the QTL IciMapping 4.1 software41. The PDS and AUDPS were used to locate QTLs for resistance. ICIM was performed every 0.2 cM with a probability in stepwise regression (PIN) of 0.001. The significant LOD threshold for QTLs of each trait was determined with a 3,000-permutation test at P = 0.05.Because several SLAF-seq markers were often mapped to the same position, in such cases only one marker was selected and used for QTL analysis. QTL analysis was conducted using the ICIM methodSupplementary Information


The assumption of a link between common symptoms such as febrile illness, diarrhea and the eruption of primary teeth has been established over many centuries. According to traditional beliefs in Ethiopia, diarrhea and fever at the time of milk teeth eruption may be due to a worm in the child’s gums. Current medical observations show little more than restlessness, drooling, and finger sucking resulting from teething. The purpose of this research was to assess mothers’ traditional beliefs and practices towards teething symptoms.A cross-sectional descriptive study design was used with the convenience sampling technique. Mothers were approached at the pediatric Out Patient Department (OPD) of Jimma University Specialized Hospital, southwest Ethiopia. A structured questionnaire was used for data collection. The data were analyzed by SPSS (version 20).A total of 107 mothers were interviewed. Ninety-eight (91.6%) claimed that teething was associated with various symptoms. Ninety-seven (90.7%) attributed diarrhea to teething. Only one mother said she would give her child Paracetamol to relieve the teething symptoms. Five (4.7%) mothers said they would allow their children to bite on a pacifier. Ten mothers (9.3%) said that they would prefer the child’s milk tooth to be extracted. Some of the practices by mothers to relieve the symptoms include rubbing the gum of the child with garlic (12.1%) or rubbing the gum with herbs (6.5%).Most of the mothers had misconceptions about the symptoms that usually appear during teething. Health education should be provided by dentists and professionals concerned with child care in correcting these misconceptions and cultural beliefs about teething symptoms.The online version of this article (10.1186/s12903-018-0619-y) contains supplementary material, which is available to authorized users. The eruption of teeth (teething) is the movement of the teeth from their pre-eruptive position in the alveolar bone through the mucosa into the oral cavity as defined by Carpenter J V in 1978 in a study that assessed the relationship between teething and systemic disturbance . Most paThe study was conducted in Jimma University Specialized Hospital (JUSH) located in southwest Ethiopia, Oromia region. Jimma is the largest city in southwest Ethiopia located 352 km away from Addis Ababa. Jimma University Specialized Hospital is one of the oldest public hospitals in the country. It was established in 1937 by Italian invaders for the service of their soldiers. It is the only teaching and referral hospital in the southwestern part of the country. It provides services for approximately 15,000 inpatients, 160,000 outpatients, 11,000 emergency cases and 4500 deliveries a year from the catchment population of about 15 million people.The Informed consent process was approved by the Jimma University’s Institute of health sciences community-based education office (CBE). Permission was also obtained from the JUSH Medical director’s office. Verbal Informed consent of the participants was obtained. The confidentiality of the information they provide was assured by removing unique identifiers from the questionnaire. The privacy of the mothers was maintained during data collection as the interview was conducted in a secluded area.The data were collected from August 10–14, 2015.Cross-Sectional study design was used..All mothers who visited JUSH’s pediatric OPD, southwest Ethiopia from August 10–14, 2015Convenience sampling technique was used. The working days of the week from August 10–14, 2015 were randomly selected. All mothers who presented at the pediatric OPD and verbally consented were interviewed.Data were collected by the interviewer-administered questionnaire which was adapted from studies done by Kakatkar et al. in India and by Awadkamil in Sudan , 13. TraIncome classification used for this study is based on the scheme of Africa Development Bank (AfDB). In this study, it is adjusted to income per month and converted to Ethiopian currency ‘Birr’ according to National bank of Ethiopia currency rate during the data collection time.Poor class: - those who earn less than 2$/dayFloating class: - those who earn 2$-4$/dayLower middle class: - those who earn 4$-10$/dayUpper middle class: - those who earn 10$-20$/dayRich class: - those who earn greater than 20$/dayp < 0.05).Data collected were analyzed by SPSS software (version 20). Descriptive statistics were used. Chi-square tests of significance (Fisher’s exact test) were used with a 95% confidence interval ((Table 1).A total of 107 mothers were interviewed. The mean age of the mothers was 29.07 (SD 9.24); Sixty-two (57.9%) were within the age range of 20–29. Sixteen (15%) of the mothers had no formal education, four (3.7%) were only able to read and write, and 24 (22.4%) had a university or college education. Fifty-three (49.5%) were housewives, civil servants were 24 (22.4%) and 6(5.6%) were professionals. Ninety-two (86.0%) live in urban. Thirty (28%) of the mothers comprised of the poor class while only Fourteen (8.4%) made up the rich class p-value = 0.031) . Forty-one (38.3%) of them believed that teeth eruption ends around 2 years of age and it was statistically significant with educational level  and place of residence  (Table (Table 2).Seventy (65.4%) of the mothers thought that teeth start to erupt around 6–7 months of age Table  and it wP-value = .024) (Table ).Most mothers (91.6%) perceived that teething was associated with various symptoms like diarrhea, fever, poor appetite, irritability, vomiting; while only 9 8.4%) thought that teething was not associated with any symptoms. Most mothers (90.7%) attributed diarrhea, (72.9%) irritability, (61.7%) poor appetite, (48.6%) fever or vomiting 35.5%) to teething . There were 60 (56.1%) who said they would not give their child anything (Table ). 36.6% claimed that non-treated teething symptoms would result in severe illness, while 7.5% thought death could happen ,which was less than the study conducted in India by Kakatkar et al. in 2010 (81.3%) [p-value = 0.006) and was less than the study in India (81.3%) [p-value = 0.019) and place of residence , which was significantly less than the study in India (76.3%) [. in 2005, where 95.2% of the mothers ascribed various ailments to teething [. in 2017) [Uti et al in 2005, found 64.0% to associate diarrhea to teething [Regarding the knowledge of mothers about tooth eruption in the current study, 65.4% believed that teeth eruption starts from 6 to 7 months of age and it differed according to mothers’ age . A Similteething . Howeverteething .This mayin 2017) . This diin 2017) . In Ibadin 2017) . The othteething .In Egyptteething .These diteething  and Egypteething , but lowteething . In our teething , but higteething , Egypt


Phenotyping was done under two environmental conditions and genotyping was carried out through the KASP SNP genotyping platform. A total of 185 and 189 individuals genotyped with 170 and 179 polymorphic SNPs were used for QTL analysis for the two populations, Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222, respectively. A total of five QTLs on chromosomes 3, 5, 6, 7, and 8 for survival (SUR) and four QTLs on chromosomes 1, 3 (two locations), and 7 for the trait seedling height (SH) across the populations and over the screening conditions were identified. Except for the QTLs on chromosomes 5 and 8, the parent with AG potential, Kalarata, contributed all the other QTLs. Among the five QTLs for SUR, the second-largest QTL (qSUR6–1) was novel for AG potential in rice, showing a stable expression in terms of genetic background and screening conditions explaining 11.96% to 16.01% of the phenotypic variation. The QTL for SH (qSH1–1) was also novel. Considering different genetic backgrounds and different screening conditions, the QTLs identified for the trait SUR explained phenotypic variation in the range of 57.60% to 73.09% while that for the trait SH ranged from 13.53% to 34.30%.Anaerobic germination (AG) is an important trait for direct-seeded rice (DSR) to be successful. Rice usually has low germination under anaerobic conditions, which leads to a poor crop stand in DSR when rain occurs after seeding. The ability of rice to germinate under water reduces the risk of poor crop stand. Further, this allows the use of water as a method of weed control. The identification of the genetic factors leading to high anaerobic germination is required to develop improved DSR varieties. In the present study, two BCThe online version of this article (10.1186/s12284-019-0305-y) contains supplementary material, which is available to authorized users. The ability of rice to germinate under water is generally low. Most modern rice varieties either fail completely to germinate under water or fail to elongate the coleoptile and develop roots and shoots for further development under a long period of oxygen deprivation, resulting in partial to complete crop failure  and NSIC Rc238 (IRRI 156), developed by IRRI. All the parents belong to subspecies indica. The growth duration of NSIC Rc222, NSIC Rc238 and Kalarata is 98, 104, and 118 days, respectively. NSIC Rc222 yields an average of 5.7 t/ha to a maximum of 7.9 t/ha. Meanwhile, NSIC Rc238 is characterized by an average yield of 6.4 t/ha to a maximum of 10.6 t/ha. Kalarata on the other hand is a low yielding landrace with yields ranging between 2 and 4 t/ha. Plant height of Kalarata, NSIC Rc 222 and NSIC Rc 238 are 144, 98 and 104 cm, respectively. A population of 205 lines for each of the crosses  underwent phenotypic evaluation. Further, genotypic profiles of a set of 189 and 185 lines were generated for the two populations, respectively, which were used in the study. The parents for each population were used as checks for the respective populations.This study was conducted using two BCBoth populations underwent two screening conditions for the evaluation of AG: one using trays filled with garden soil placed on a concrete screening table and the other directly on the puddled soil of a screenhouse, providing more natural field conditions for the experiments. Controlled experiments were also conducted under dry direct-seeded conditions in a screenhouse Fig. . The expDry seeds were used for the experiments. Dormancy was broken by keeping the seeds at 52 °C for 72 h in a hot-air oven. A 15X15 α-lattice design with three replications was used to conduct the experiments in all conditions.3) filled with fine garden soil were marked with a grid marker maintaining seeding depth of 1.0 cm and 15 lines per tray. Thirty seeds from each entry were sown in each line. After seeding, the lines were covered with fine garden soil. The trays were submerged carefully in concrete benches filled with 7–8 cm of water from the soil surface of the trays. This water depth was maintained for the entire experiment duration (21 days). Two measuring scales were used for each table to constantly monitor and maintain the water level.For the table screening, seedling trays  was determined as well. For the experiment under aerobic conditions, seedlings were also counted on the 14th and 21st day after seeding (DAS) to determine germination percentage (GER) and the height of five random seedlings were measured and averaged to determine the mean SH for each line. Additionally, data on seed pericarp color was collected for both populations after dehulling the seeds of each line. This was done through visual scoring where the line is scored as 1 if 100% of the seeds are white, 3 if 75% of the seeds are white, 5 is 50% of the seeds are white, 7 if 25% of the seeds are white, and 9 if all seeds are red. The subset of lines scored with 1 was also used for downstream analyses apart from the two individual population analyses.http://bbi.irri.org/products). The linear mixed model described below was used for α-lattice design analysis:SUR was calculated as the percentage of the number of surviving seedlings relative to the total number of seeds used. Analysis of variance (ANOVA) was conducted using PBTools V 1.4.0. and CropStat7.2.3 , the effects of replications and blocks within replications were considered as random, whereas, for the computation of variance components, the effects of genotypes, blocks, and replication were considered random. Broad-sense heritability was calculated asP2 stands for broad-sense heritability, Here, HTaq polymerase, free nucleotide, and MgCl2 in an optimized buffer. End-point fluorescence data were visualized with a microplate reader  and analyzed by Klustering Caller software7. Reaction mixtures consisted of final volumes of 5 μL containing 2.5 μL of 2 X KASP V4.0 Mastermix, 0.056 μL of assay primer mix , and 50–100 ng of genomic DNA. An S1000 Thermal Cycler (Bio-Rad) was used with the following cycling conditions: 94 °C for 15 min, 9 cycles of 94 °C for 20S, touchdown starting at 65 °C for 60S (decreasing 0.8 °C per cycle), 32 cycles of 94 °C for 20S, and 57 °C for 60S.A total of 189 families from the Kalarata/NSIC Rc222 population and 185 families from the Kalarata/NSIC Rc238 population were selected for genotyping. Leaf samples were collected from a bulk of 20 plants per family and lyophilized for DNA extraction. Genotyping was done through the KASP assay genotyping platform of 1100 SNPs based on MSU Rice Genome Annotation Project Release 7  presence of the detected QTLs in varying combinations; (2) percentage survival under anaerobic conditions; (3) white pericarp color; and (4) percentage germination under anaerobic conditions.Two populations along with their respective parents as checks were analyzed for their phenotypic performance under stress and non-stress conditions. Significant differences were observed among the genotypes while no significant difference was observed between replicates. Table Continuous variation with a small skewed frequency distribution was observed for all the traits under study. A significant positive correlation was observed for SUR across screenhouse and tray-on-table screening conditions for both 14 and 21 DAS. Higher correlation was observed within each screening conditions compared to those across screening conditions for the Kalarata/NSIC Rc238 population  showed a LOD score of 15.30 to 20.31 and explained 31.58% to 39.66% of the phenotypic variation, with an additive effect of 17.15% to 24.28%, followed by qSUR6–1 , which explained 14.28% to 16.01% of the phenotypic variation, detected by a LOD score of 5.97 to 6.96, and had an additive effect of 9.79% to12.72%. qSUR3–1  contributed 11.04% to 12.79% of the phenotypic variation, with an additive effect of 10.85% to 14.03%. The SUR QTLs qSUR5–1  and qSUR8–1  from high-yielding susceptible parent NSIC Rc238 contributed 9.76% to 10.82% and 11.28% to 12.04% of the phenotypic variation, with LOD scores of 4.10 to 4.57 and 4.78 to 5.21, respectively , which is consistent over both screening conditions. It explained a phenotypic variation of 12.5% to 22.1%, with a LOD score of 5.4 to 9.4. The second largest effect QTL detected for SH was consistent with SUR QTL qSH3–1 , which was also consistent in both screening conditions. This QTL explained phenotypic variation of 10.1% to14.4%, with a LOD score of 3.7 to 5.8 and an additive effect of 0.84% to 5.17%. QTL qSH7–1Rc238-SCR-21 was identified with a LOD score of 4.6 and explained 10.7% of the phenotypic variation and had an additive effect of 4.57%. It was identified only under screenhouse conditions and was located in a different position from the identified QTL (qSUR7–1) for SUR  was detected for SH with an LOD score of 6.1 that could explain 14.1% of the phenotypic variation  was identified by a LOD of 12.07 to 14.10 explaining 25.80% to 29.49% of the phenotypic variation with an additive effect of 20.01% to 24.52%. The second largest effect QTL qSUR6–1  explained 11.96% to 13.33% of the phenotypic variation and was detected by a LOD score of 5.18 to 5.81 and had an additive effect of 10.30% to 11.95%. The QTL qSUR3–1  explained 9.49% to 11.47% of the phenotypic variation with an additive effect of 9.27% to 12.31% and was detected by a LOD score of 4.15 to 4.93  identified by a LOD score of 5.18 to 7.17 had an additive effect of 1.3% to 3.4% and could explain 13.5% to 18.5% of the phenotypic variation. A second position on chromosome 3, qSH3–2 (named as qSH3–2Rc222-SCR-14 and qSH3–2Rc222-SCR-21) that is different from the detected QTL from the other population for the same trait was identified by an LOD score of 3.78 to 4.87 and could explain 10.2% to 11.7% of the phenotypic variation , which was contributed by susceptible parent NSIC Rc222 (Table qSH1-2Rc238-CON-21) was also detected in a different position in control conditions from the one detected under stress. This QTL was detected by a LOD score of 10.16 and explained 21.9% of the phenotypic variation with an additive effect of 2.3%  with a large and stable effect across the two populations were identified in this study. We conducted a combined analysis of the two populations to understand the effect of varying QTL combinations on germination under aerobic and anaerobic conditions at 21 DAS. Figure qSUR7–1 showed the highest effect on the trait, followed by qSUR6–1 and qSUR3–1, respectively. It was also observed that lines with qSUR3–1 and qSUR6–1 had a significantly higher advantage over the lines with only one of the two QTLs. The QTL qSUR7–1, however, provided a high advantage even without the other two QTLs. Its combination with qSUR6–1 increased the total advantage, which was further increased in the lines with all three QTLs combined, although these are not statistically significant. The increase in advantage of a QTL when combined with the other QTLs indicates their additive nature. Lines with the combination of qSUR7–1 and qSUR3–1 were not found in both populations. Moreover, lines without the three QTLs had the lowest germination of all classes which was lower than the population mean.Three QTLs  was also detected near the said loci at 60 cM and 62 cM for Kalarata/NSIC Rc222 and Kalarata/NSIC Rc238 populations, respectively  and qSUR6–1  were found significant in the Kalarata/NSIC Rc222 population for survivability at 14 and 21 DAS  was significant for survivability for the Kalarata/NSIC Rc238 population  used in this population has a red-colored pericarp. In the past, studies have detected QTLs for high anaerobic germination at chromosome 7, which lie closely to the We also identified a set of eight lines with different combinations of the three major QTLs identified in this study, white-colored pericarp and high germination under aerobic and anaerobic conditions Table . These lIn this study, we aimed to identify novel and robust genomic regions from the landrace Kalarata that can potentially contribute to further increasing the anaerobic germination potential of existing direct-seeded rice varieties. Two populations derived from the cross of Kalarata with elite recipient lines NSIC Rc222 and NSIC Rc238 were developed and evaluated for germination under different anaerobic screening conditions to identify QTLs stable across genetic backgrounds and across screening methods. In the past, several phenotyping protocols have been considered for screening rice lines for AG. Studies have shown that some rice genotypes can germinate under water and elongate their coleoptile to some extent but ultimately fail to develop roots and shoots. The ability to germinate under water and successfully develop roots and shoots is referred to as SUR. This innate phenomenon is the key trait to mark the genetic variation among the genotypes for several underlying traits that play a role in successful germination under water. Together with SUR, SH is the second most important trait because it reflects fast coleoptile elongation, which is frequently used to study the genetics of anaerobic germination. In our study, the two traits were recorded at 14 and 21 DAS in controlled tray-on-table screening as well as under more field-like conditions in the screenhouse. Apart from this, germination in a controlled environment was also used as a trait to determine early and uniform emergence under non-stress conditions Fig.  also obsqSUR3–1), chromosome 6 (qSUR6–1), and chromosome 7 (qSUR7–1) across both populations and both screening conditions, explaining a stable expression of phenotypic variation ranging from 47.34% to 64.98%. qSUR7–1 was the largest effect QTL for SUR, followed by qSUR6–1 and qSUR3–1, respectively. One highly significant QTL on chromosome 1 (qSH1–1) for SH was detected in both populations and screening conditions, explaining 12.5% to 22.1% of the phenotypic variation for seedling height. Altogether, the QTLs for the trait SUR explained phenotypic variation ranging from 47.34% to 87.78% while for trait SH it ranged from 10.1% to 36.5%. The other QTLs identified in this study were either population specific or screening condition specific . This trait is crucial not only for DSR and AG but also for stresses such as stagnant flooding, for which taller SH is a desirable trait.The results of the QTL analysis revealed three highly significant QTLs for SUR Fig.  on chromqSUR7–1 was the largest QTL identified in this study. This QTL co-locates with the Rc gene, which is responsible for pericarp color in rice. In the study, a QTL for pericarp color was also identified which coincided with the known position of the Rc gene at 6.07 Mb of chromosome 7  under anerobic condition. Table S2. List of QTLs detected for seedling height in the BC1F2:3 population of Kalarata and NSIC Rc238. Table S3. List of QTLs detected for seedling height in the BC1F2:3 population of Kalarata and NSIC Rc222. Table S4. List of QTLs detected for pericarp color in the BC1F2:3 mapping populations of Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222. Figure S1. Line graph showing the water temperature profile during the experiment period in screen house and on Tray conditions. Figure S2. The QTL likelihood curve of LOD score showing peak marker and confidence interval for the trait 21 DAS survivability in Kalarata/NSIC Rc238 population in screenhouse screening conditions. The green line indicates confidence interval while horizontal lines indicate the significant logarithm of odds threshold at 95% and 99% confidence levels (from the bottom to the top) based on 10,000 permutations. The figures on the right show the effect of each of the peak markers. All QTLs are contributed by tolerant parent, Kalarata. Figure S3. The QTL likelihood curves of LOD score showing peak marker and confidence interval for the trait 21 DAS seedling height for (A) Kalarata/NSIC Rc238 and (B) Kalarata/NSIC Rc222 in screenhouse screening conditions. The green line indicates confidence interval while horizontal lines indicate the significant logarithm of odds threshold at 95% and 99% confidence levels (from the bottom to the top) based on 10,000 permutations. All QTLs are contributed by tolerant parent, Kalarata. Figure S4. Frequency distribution of the survival (SUR) trait for Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222 populations under different screening conditions (screenhouse and tray-on-table) and data collection periods (14 DAS and 21 DAS). Dotted lines refer to the susceptible parent (NSIC Rc 238/NSIC Rc 222) while solid lines refer to the tolerant parent . Figure S5. Relation between grain pericarp color and survival under anaerobic condition. 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Cell culture is a ubiquitous and flexible research method. However, it heavily relies on plastic consumables generating millions of tonnes of plastic waste yearly. Plastic waste is a major and growing global concern. Here we describe a new cell culture dish that offers a culture area equivalent to three petri dishes but that is on average 61% lighter and occupies 67% less volume. Our dish is composed of a lid and three thin containers surrounded by a light outer shell. Cell culture can be performed in each of the containers sequentially. The outer shell provides the appropriate structure for the manipulation of the dish as a whole. The prototype was tested by sequentially growing cells in each of its containers. As a control, sequential cultures in groups of 3 petri dishes were performed. No statistical differences were found between the prototype and the control in terms of cell number, cell viability or cell distribution. Plastic waste is a major and growing concern around the world. The oceans alone receive an estimated 1.15 and 2.41 million tonnes of plastic waste yearly . In 2014Cell culture is a flexible tool that can be performed in a variety of dishes and surfaces for diverse types of applications such as drug testing, biomarker identification and expression of recombinant proteins –14. In c2 , left resting for 4 days in a clean environment, submerged in 70% ethanol for 1 day and finally left to dry for 1 day in a sterile environment. The lid used in the prototype is the lid of a petri dish 100 mm in diameter (430167—Corning Inc.).The outer shell was 3D printed (Ultimaker-2—Ultimaker) with a 2.85 mm, transparent polylactic acid (PLA) filament (1614—Ultimaker). PLA is one of the most commonly used polymers for 3D printing, is biocompatible and is used for biomedical and packaging applications . The layHeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) media (41966–029—Gilbco), 10% fetal bovine serum (FBS) (10270–106—Gilbco) and 1% Penicillin-Streptomycin (15140–122—Gibco). 0.6 million cells were seeded into the first layer of the prototype. After 72 h the cells were trypsinized, washed and a sample stained with trypan blue (T8154—Sigma) 1:1. The cells were analyzed for cell number and percentage of live cells in a TC20 automated cell counter (Bio-Rad Laboratories) according to the manufacturer’s instructions. The first layer, now empty, was removed with the help of a sterile tweezer. From the cells grown in the first layer, 0.6 million cells were seeded into the second layer and the process was repeated. As a control, the same procedures were conducted in three consecutive 100mm in diameter petri dishes (430167—Corning Inc.). In total, six prototypes and six groups of three petri dishes were used to assess cell number and percentage of live.Cells were cultured as described above. After 72 h, the media was discarded, the cells quickly rinsed in phosphate-buffered saline (PBS) solution (pH 7.4) and fixed in 4% formaldehyde (1.04003.2500 –MilliporeSigma) in PBS (pH 7.4) for 20 min at RT. Then, the cells were permeabilized in 0.01% triton in PBS (pH 7.4) for 15min at RT, blocked in 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) solution (pH 7.6) for 1h at 4°C and incubated ON at 4° C with a FITC anti-beta actin antibody (ab6277—Abcam) 1:100 in 3% BSA in TBS (pH 7.6). The cells were washed 3 times for 5 min in PBS (pH 7.4) at RT and incubated for 5 min with Hoechst 33342 (ab228551—Abcam) 1:5000 in PBS (pH 7.4) at RT. Finally, the cells were washed 3 times for 5 min in 0.1% Tween in PBS (pH 7.4) at RT and rinsed 1 time in PBS (pH 7.4) at RT.2. A set of 15 x and y coordinates were randomly generated and used for each dish. At each random location, fluorescence imaging was carried out using a Nikon Plan Fluor 10x/0.3 objective with a Lumencor SOLA SE II Solid State White Light Engine for illumination along with Semrock LED-DAPI-A-000 and GFP-3035D-000 single-band filter sets. Images were captured and saved using an Andor Zyla 5.5 sCMOS USB3 camera and the number of nuclei present in each image was counted using CellProfiler [2 was calculated based on the size of each image (1.011 mm2).Images of stained cells on the control petri dishes and prototypes were acquired using a Nikon Ti-E Eclipse microscope with a Prior H117 motorized stage, controlled using Micro-Manager. 3 petriThe weight was assessed in six individual control petri dishes (430167—Corning Inc.) with their respective lids. The weight of each petri dish was multiplied by three, since one prototype has a culture area equivalent to three petri dishes. The weight of six prototypes was also assessed, where each prototype was composed of a lid, three layers and one shell. Since the dishes and prototypes are cylinder-shaped, their external volume was estimated based on radius and height.2. Unpaired t-test was used to analyse the results of: dish weight and dish volume.One-way ANOVA was used to analyse the results of: cell number, percentage of live cells and number of cells per mmThe prototype is composed of a lid, three culture layers and a structural outer shell . The cul2  plastic containers. Our prototype, which weighs on average 20.0 grams, has a culture area equivalent to three petri dishes but three petri dishes weigh on average 51.4 grams. Thus, the prototype is 61% lighter than the control . The dif3 but, as described above, it has a culture area equivalent to three petri dishes, which have a combined average volume of 434.0 cm3. Hence the prototype is on average 67% more compact than the control, a difference that is highly significant  (The prototype is on average 142.8 cm 0.0001) . A less S1 Table(XLSX)Click here for additional data file.


Cutoff values for lean mass and muscle strength are still controversial in the diagnosis of sarcopenia. The use of European, American and Asian consensus outside these regions may lead to important diagnostic errors. We hypothesized that there are significant differences between the cutoff points from Brazil and Europe in older people. This is a cross-sectional analyses of 502 older adults from SARCOS study, conducted at São Paulo - Brazil. All subjects underwent DXA analyses of total body. Lean mass was obtained from appendicular lean mass by height2 and muscle strength by dynamometer of dominant hand. The Brazilian cutoff points were based on 25th percentile by gender. The European ones were from EWGSOP 2. Agreement was assessed by the Kappa coefficient. The mean age was 78.39 ± 7.08 years old and 277 (55.18%) individuals were women. Among the ethnic groups, 339 (67.53%) were caucasian, 145 (28.88%) afrodescendants and 18 (3.59%) asians. The Brazilian cutoffs for muscle strength were 26 kg for men and 16 kg for women ; while those for lean mass were significantly lower, 6.56 kg/m2 vs. 5.56 kg/m2, respectively. The prevalence by EWGSOP 2 was higher than that obtained by the Brazilian cutoff points , even though these criteria presented Kappa = 0.792; p <0.001. Considering these disparities, 6 out of 100 subjects are considered sarcopenic by European criteria and not by the Brazilian cutoffs. There are significant differences in sarcopenia cutoffs between Brazil and Europe, and this cause important diagnostic variations.