Background: A current popular aesthetic problem, especially among younger women, is striae distensae (SD), also referred to as “stretch marks.”. Aim: The potential use of the 675 nm laser has been investigated in the treatment of SD. Methods: Patients underwent three sessions of the 675 nm laser with a 1-month interval between sessions. A total of three sessions were performed. The Manchester Scar Scale was used to assess stretch mark changes, and the mean scores related to each parameter at baseline and 6M FU after the last treatment session were measured. A clinical photographic evaluation was performed to show the aesthetic improvement of SD. Results: The patients’ treated areas were the abdomen, thighs, buttocks, and breasts. Mean scores related to each Manchester Scar Scale parameter, with their relative percentage change, at baseline and 6M FU after the last treatment session were significantly improved. The total mean Manchester Scar Scale score significantly diminished from 14.16 (±1.30) to 10.06 (±1.32) at 6M FU (p < 0.01). The clinical photographs showed promising aesthetic SD improvement. Conclusions: 675 nm laser therapy demonstrated a good tolerance for the treatment of stretch marks applied to various body areas preventing any discomfort for the patient and with a significant improvement in skin texture. Striae distensae (SD), also known as “stretch marks”, are a common aesthetic problem, especially in young women. Clinically characterized by atrophic linear lesions, first reddish, then porcelain-white, they are mainly localized on the abdomen, breasts, and thighs in adolescents, they are common on the thighs, buttocks, breasts , and back  . SDs genGenetic predisposition in association with hormonal changes, drugs, and mechanical stress contribute to SD occurrence, but the exact pathogenesis remains unclear. The main hypothesis is structural damage of dermal collagen and elastic tissue. In fact, an alteration of the architecture of the dermal connective tissue, with the involvement of the extracellular matrix (ECM), has been demonstrated along with damage to fibrillin, elastin, fibronectin, and collagen fibers. Elastases released from mast cells and macrophages seem to be responsible for the initial structural dermal alterations followed by a reorganization of collagen and fibrillin. The degradation of collagen finally results in atrophic scarring.Recently, the 675 nm wavelength showed successful results in facial aging ,4,5 as wA proven effect of this laser system has been reported also for facial rejuvenation in Asian population . From thThis study’s goal was to assess the clinical effectiveness of a diode laser with a wavelength of 675 nm  for the non-invasive treatment of stretch marks (Striae distensae) reduction on the patient’s abdomen, thighs, buttocks, and breasts. Specifically, a total of 6 abdomens, 6 thighs, 7 buttocks and 13 breasts were treated. The laser device is a continuous (CW) diode laser that emits a wavelength of 675 nm, and it is equipped with a 13 × 13 mm scanning system, which can be accessorized with contact and temperature sensors integrated into the handpiece and is able to generate fractional micro-zones (DOT) of 0.7 mm width of either sub-ablative and selective thermal damage on the skin. Each DOT receives radiation from the CW source via the Power and Dwell time parameters. By adding a distance (Spacing) between each DOT, the scanning system enables frac-tionally uniformly covering the treatment area. This preserves the epidermal layer thanks to an integrated skin cooling system, protecting skin health while minimizing downtime. Each emission may reach a thermal column depth of more than 1 mm. Before starting the study, informed consent was obtained from all the participants. A total of 32 female patients with SD, ranging in Fitzpatrick skin phototype from I to IV  and with a mean age of 31 (±7.5), were enrolled. Study exclusion criteria included: (1) hypersensitivity to light in the red and near-infrared wavelength region; (2) use of photosensitizing drugs, anticoagulants, and/or immunosuppressants; (3) photosensitivity diseases; (4) pregnancy; (5) personal or family history of skin cancer, (6) sun exposure in the previous month; and (7) the presence of tattoos or skin disorders on the areas to be treated.Patients were treated with 3 sessions of the 675 nm laser using a conductive gel  with a 1-month interval between sessions. Areas to be treated were cleaned with a mild soap, rinsed with water, and topical anesthetic was applied before the session started. Based on the subject’s skin type and level of tolerance, the energy treatment assessment was performed for each patient on a specific area “test.”. Within 48–72 h, the test’s outcome was noticeable. Mild erythema was recognized as the endpoint. Treatment was administered by softly passing the handpiece over the skin’s surface, within spots close to each other without overlapping but without leaving untreated areas. Following treatment gauzes soaked in cold water were used to cool the skin.The Manchester Scar Scale  was usedAdverse events such as edema, burns, formation of keloids, excessive pain, crusting, scars, prolonged erythema, blisters, or dyschromia were evaluated at the post-treatment visit.t-test was used to test all the outcome data for statistical significance with the SPSS program version 25.0 . The level of significance for statistical tests was (p < 0.01). Data were represented as means ± standard deviation (SD).Paired Student’s p < 0.01) showing excellent results as reported in p < 0.01)  . The cliStretch marks’ treatment is still difficult even though several therapeutic options are currently available. Accordingly, the available treatments for SD work mainly on collagen synthesis, but despite a multitude of options, no single treatment has yet proven effective . They inThe energy delivered to the collagen can induce regeneration, promoting the production of dermal collagen, and the straightening of elastic fibers. Indeed, the newly created thermal column diffuses heat to the surrounding areas causing immediate shrinkage and denaturation of the collagen with subsequent new collagen formation through the stimulation of fibroblasts and neocollagenesis activation.This diode laser with a wavelength of 675 nm generates 1 mm microzones of thermal damage that, thanks to the cooling and selectivity of the dermal layer, do not damage the epidermal layer. Thanks to the increased focus on small spots, microscopic necrotic epidermal debris or dermo-epidermal detachment, which is typical of the post-operative course of Near Infrared (NIR) systems, were not observed. Our study findings pointed to a notable SD improvement for all patients, as well as a significant improvement in finish, texture, color, distortion, and contour, along with a significant increase in skin elasticity as clearly shown in the photographic evaluation. Due to the preventive cooling of the skin, the procedure is also relatively painless. The treatment is simple to administer, non-invasive and did not have reported side effects or complications.Limitations include the limited number of patients and the lack of a histologic investigation.The efficacy of 675 nm laser for treating stretch marks on the abdomen, thighs, buttocks, and breasts showed good tolerance with a significant improvement in skin texture and quality of stretch marks, and temporarily and minimally restricts the patient’s daily activities preventing any discomfort for the patient.


We observed a significant decrease in richness and diversity and an increase in dominance over time . The oral microbiome is highly dynamic during the first weeks of life, and beta diversity suggests a microbial succession in early life. The introduction of oral diet does not change the community structure, but affects the abundance, especially of Streptococcus. Our results indicate that although time is related to significant changes in the oral microbial profile, oral feeding benefits genera that will remain colonizers throughout the host’s life.Very low birth weight (VLBW) infants, mostly preterm, have many barriers to feeding directly from the mother’s breast, and need to be fed alternatively. Feeding is a major influencer in oral microbial colonization, and this colonization in early life is crucial for the promotion of human health. Therefore, this research aimed to observe the establishment of oral microbiome in VLBW infants during their first month of life through hospitalization, and to verify the impact caused by the implementation of oral diet on the colonization of these newborns. We included 23 newborns followed during hospitalization and analyzed saliva samples collected weekly, using  Low birth weight LBW, <2500 g) is one of the main causes of neonatal mortality and morbidity in low- and middle-income countries, along with preterm birth, infections, and asphyxia at birth . This te500 g is Most VLBW infants are premature, and enteral feeding is often a challenge, due to their immature physiological system and neurological development , 4. ThusStreptococcus salivarius [Streptococcus species , which livarius , and oncucleatum , can inducleatum , oral caucleatum , and sysucleatum .Staphylococcus aureus, as described in the study by Underwood and Sohn [In addition to all the challenges preterm newborns face, they are also more susceptible to being colonized by oral pathogens, such as methicillin-resistant and Sohn , which cand Sohn , 16, sucand Sohn  and expoand Sohn .After birth, some variables can affect the oral microbiome in very low birth weight infants, for example, the delivery mode . Also, pDue to their prematurity, VLBW infants stay in NICUs for a long time, and need to be fed by alternative routes until oral feeding is possible. Therefore, the purpose of this study was to evaluate the establishment of oral microbiome in VLBW infants during their first month of life through hospitalization in a NICU, considering the role of oral diet implementation.The study was approved by the Research Ethics Committee of the Hospital das Clínicas of the University of São Paulo  (CAAE: 09673619.7.0000.0068) and the newborns’ parents were informed about the study during recruitment and signed a written informed consent form stating their agreement with the research.This is a longitudinal, observational study, which included VLBW infants hospitalized in the NICU of Hospital das Clínicas of the University of São Paulo . We collected weekly samples from 23 preterm babies born with very low birth weight, who were hospitalized in a NICU for at least four weeks. All newborns included were delivered by cesarean section and received oral diet at some point during hospitalization, which consisted of expressed human milk or milk-based infant formula for preterm. The chances of death in early life increases as lower the birth weight and gestational age of the infant . The samAll saliva samples were collected using two sterile swabs, rubbing them in the baby’s oral cavity for about 30 seconds, covering the cheeks and tongue. The first sample was collected within the first 24 hours after birth, and subsequent collections were carried out weekly. For this study, we selected samples collected in the first, third and fourth postpartum weeks, and in the weeks immediately before and after the implementation of oral diet. After collection, the cotton swabs were transferred to a 15-ml tube containing 500 μl of Phosphate-Buffered Saline  and stored at -20°C until shipped, within 24 hours after collection, to the Molecular Microbiology Laboratory, where they were stored at -80°C until DNA extraction was performed.Initially, the samples were pre-treated as described by Cortez et al. , which b16S rRNA region amplified by PCR technique was V3-V4, with the primers 5′- CCTACGGGNGGCWGCAG-3′ forward and 5′-GACTACHVGGGTATCTAATCC-3 reverse to the described region [2. The MiSeq platform was used for image analysis, base calling, and data quality assessment.The d region , with Il16S rRNA amplicons were analyzed using software QIIME2 version 2021.11  [qiime2R [phyloseq [microbiome [vegan [Ecology) . ChimeriEcology) , with boEcology)  and the Ecology) , 29. Div[qiime2R , phylosephyloseq , microbicrobiome , and vege [vegan  in the Re [vegan . In thise [vegan  , by observing samples over time  and in relation to the introduction of oral feeding (before and after). Relative abundance of the representative genera and alpha diversity were analyzed by linear model regression using MASS  and jtoo [jtools  R packag package ) was perxHeatmap , and the package ) with a  package . All graage in R .The newborns were assigned to a single group for analysis, which consisted of observing the effect of time on microbial colonization, and how diet could affect the oral microbiome of these babies. For this purpose, a total of 23 VLBW infants were included, with samples collected in the first (n = 19), third (n = 21) and fourth (n = 18) postpartum weeks, and also in the week before (n = 22) and the week after (n = 23) the implementation of oral diet, totaling 89 samples. For some babies, the sample from weeks before and after the implementation of oral diet coincided with the third or fourth hospitalization week. The variation in the number of samples during follow-up is due to some loss during the experiment conduction, since some samples did not have sufficient DNA concentration or did not amplify in the PCR step.All newborns enrolled in this study were preterm, with a mean gestational age of 30.52 weeks  , and a mStreptococcus, Staphylococcus and Enterobacteriaceae members, by the third week of life. In the fourth week of life, when most babies had already started oral diet, the dominance of Enterobacteriaceae decreased and Streptococcus and Staphylococcus remained dominant (Streptococcus (10.86%), Staphylococcus (9.59%), Escherichia-Shigella (6.23%), Enterobacter (5.74%), and Acinetobacter (3.84%). Over time, there was an increase in the relative abundance of Staphylococcus (32.64%), Streptococcus (23.62%), Enterobacter (9.00%), Klebsiella (6.38%), Haemophilus (4.86%), Gemella (3.93%), and Neisseria (3.33%), and a slightly decrease in the abundance of Escherichia-Shigella (5.35%) and Acinetobacter (3.63%). In the fourth postpartum week, Streptococcus (33.90%) became the most abundant genus in babies’ oral microbiome, followed by Staphylococcus (23.07%), Veillonella (10.91%), Enterobacter (9.36%), Haemophilus (7.47%), Klebsiella (4.02%), Neisseria (3.00%), and Gemella (2.17%). The genera Acinetobacter (0.69%) and Escherichia-Shigella (0.13%) along with Staphylococcus, had a greater decrease when compared to the third postpartum week . Other genera showed a decrease in abundance after introduction of oral diet, as Staphylococcus (28.68% to 12.38%), Neisseria (10.18% to 2.69%), Haemophilus (6.70% to 2.16%), Klebsiella (3.79% to 3.73%), Gemella (5.58% to 1.70%), Enterobacter (3.33% to 0.09%), and Clostridium_sensu_stricto_1 (1.26% to 0.01%), as shown in By assessing the oral microbiome of VLBW infants right after the implementation of oral diet, an increase in ominance  was founWhen observed the taxa composition at Amplicon Sequencing Variants (ASV) level, we found ASVs that were significantly associated with the postpartum weeks. In addition to the genus abundance results, there is a decrease of the diversity over time, mainly from the first to the further weeks of life, with a slightly increase in the abundance of the ASVs of bacteria described as oral eobiont colonizers , but thiThe alpha diversity analysis, regarding postpartum weeks, showed that the oral microbiome is richer and more diverse in the first week, and that richness and diversity, as measured by Chao1  . HoweverStreptococcus’ ASVs (19.00%). In addition, 121 ASVs were shared among all the first four weeks of life, and the most prevalent ASVs were of oral eobiont microorganisms, such as Streptococcus, Staphylococcus, Neisseria and Haemophilus. The main ASV shared in the first month  is from the Streptococcus genus, with ASVs of the Streptococcus salivarius species being the most prevalent among them.The shared ASVs diagram  shows whVeillonella and Streptococcus. 164 ASVs were common both before and after the oral diet introduction, and it was also consisted of microorganism described as oral colonizers, mainly from the Streptococcus genus , similar to the third postpartum week. After the oral diet, there is the introduction of 233 ASVs, mostly of oral eobiont colonizers such as us genus .This research evaluated the process of colonization of the oral microbiome in preterm VLBW infants in a NICU environment at different times, from birth to the implementation of oral diet, to observe the establishment of the oral microbiome. We emphasize here the decrease of genera associated with healthcare-associated infections through the weeks, and an increase in oral eobiont colonizers, especially after oral diet introduction. Also, there is a decrease in diversity and richness over time in contrast with an increase in dominance, according to alpha diversity indices, and the same was observed after oral diet implementation, except for richness. Structural changes in microbial communities were also observed, mainly driven by the decrease of abundance of some dominant genera, as seen in weighted Unifrac analysis, in both oral diet introduction and time variables.Streptococcus, Staphylococcus and Enterobacteriaceae members, while Acinetobacter, Haemophilus and Neisseria showed lower relative abundance. In hospitalized premature newborns, microbes present in the hospital environment were also found as oral colonizers, such as Escherichia, Staphylococcus, and Streptococcus [Acinetobacter, Haemophilus, and Neisseria in lower relative abundance colonizing the environment. In our study, we found similar bacteria in the babies’ mouth, suggesting that these genera may be acquired from an environmental source. However, to state this more categorically, a sampling of the environment would be necessary.In the delivery room, the newborn’s mouth is exposed to a diverse load of microorganisms, which are provided by breathing, and interaction with parents and hospital staff . During tococcus . The autStreptococcus, Veillonella, and Haemophilus. In addition, there was a decrease in genera such as Staphylococcus, Acinetobacter, Escherichia-Shigella, and Klebsiella, described as colonizers of the hospital environment [In the relative abundance transition between the first and the third weeks of life, there was a decrease in microbial diversity and richness, and this data is supported by the significant differences observed in Shannon and Chao1 indices over the weeks. This finding is associated to a microbiome shift related to the increase of oral eobiont genera, such as ironment , 47. Thiironment .Staphylococcus, Klebsiella, and Enterobacter, and an increase in the abundance of Veillonella and, mostly Streptococcus, which becomes the dominant genus when evaluating the genera relative abundance before and after the implementation of oral diet. The individual graph of genera relative abundance over time and in relation to diet status shows that oral microbiome in early life is widely dynamic and with greater inter-individual variations. However, the oral microbiome tends to acquire a certain stability over time, shifting to a more similar pattern between individuals after the implementation of oral diet.Newborns’ microbial communities evolve as the baby grows  and thisStreptococcus genus have genes encoding proteins associated with carbohydrate metabolism, specifically maltosaccharides [malP encodes the enzyme maltodextrin phosphorylase [malE gene described by the authors as an important factor for oropharyngeal colonization by Streptococcus [Most of the oral diet given to the babies was preterm infant formula, in which maltodextrin is one of the most abundant carbohydrates. It is described that some species of the charides . For exahorylase , which phorylase . Also, ttococcus .Streptococcus. Cortez et al. [Streptococcus relative abundance around the 21st day of life, slightly higher compared to the standard care group. Although we also observed an increased relative abundance of Streptococcus, this occurred around the 5th week of life, suggesting that colostrum administration favors an early increase in the abundance of this genus.Over the weeks, genera described as colonizers of the human oral cavity appear and stand out in relation to the others , 52, mosz et al. , observeStreptococcus salivarius [Streptococcus genus in newborns’ oral cavity [Streptococcus salivarius in both analyzes. This specie will serve as an adhesion site for subsequent colonizers [Streptococcus species can compete with pathogenic species for substrate, as Porphyromonas gingivalis, to prevent their adhesion and inhibit their growth [Oral microbiome colonization begins with species able to adhere to epithelial cells and salivary proteins, such as livarius , also knl cavity . We founlonizers , which slonizers , thus prr growth , 55.Lactobacillus, a genus associated with oral colonization in neonates fed exclusively or partially with human milk. This genus has properties that can inhibit the growth of pathogenic Streptococcus species, such as cariogenic Streptococcus mutans [Since most babies in our study were not breastfed, we cannot state how human milk could impact the formation of the oral microbiome in these VLBW infants. However, we found a lack of important microorganisms, such as s mutans . Additios mutans , besidess mutans .As limitations of our study, we can point out that the small sample size, along with the loss of some follow-up samples, impacted the statistical results. The sequence methodology used (16s rRNA gene sequencing) also is a limitation, as it does not allow us to go beyond the genus level for many microorganisms. In addition, the low number of babies fed with human milk did not allow us to attribute differences in the microbial profile to the intake of human milk. Also, all included infants were caesarean born, therefore our results cannot be generalized to the entire population, especially vaginally born VLBW infants. However, as strengths of our study, we can point the follow-up of the infants in different time points and the sampling before and after the oral diet implementation, which allowed us to compare how this type of diet could impact in the microbiome establishment in the oral cavity.All these findings highlight the importance of stimulating oral feeding as early as possible in VLBW infants, mainly through an oropharyngeal colostrum administration protocol, which is very well described for its safety, simplicity and potential beneficial effects, with no additional risk to infants . Many stStreptococcus relative abundance.In conclusion, our results suggest that, although time is related to significant changes in the oral microbial profile of very low birth weight babies, the implementation of oral diet also plays a major role in the initial establishment of microbiome, favoring specific genera that will remain as oral eobiont colonizers throughout the host’s life, as shown with S1 FigPrincipal coordinate analysis (PCoA) for (A) unweighted  and (B) weighted  Unifrac distance metrics over time  and according to the oral diet status (before and after).(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table#p-value was based on linear model test, and q-value results were confirmed with False Discovery Rate (FDR) post-hoc.(DOCX)Click here for additional data file.S5 Table#p-value was based on linear model test, and q-value results were confirmed with False Discovery Rate (FDR) post-hoc.(DOCX)Click here for additional data file.S6 Table#p-value was based on a linear model test, and q-value results were confirmed with False Discovery Rate (FDR) post-hoc. * q-value was considered significant when ≤ 0.10.(DOCX)Click here for additional data file.S7 Table#p-value was based on a linear model test, and q-value results were confirmed with False Discovery Rate (FDR) post-hoc. *q-value was considered significant when ≤ 0.10.(DOCX)Click here for additional data file.


Claudin-low breast cancer (CLBC) is a subgroup of breast cancer discovered at the molecular level in 2007. Claudin is one of the primary proteins that make up tight junctions, and it plays crucial roles in anti-inflammatory and antitumor responses as well as the maintenance of water and electrolyte balance. Decreased expression of claudin results in the disruption of tight junction structures and the activation of downstream signaling pathways, which can lead to tumor formation. The origin of Claudin-low breast cancer is still in dispute. Claudin-low breast cancer is characterized by low expression of Claudin3, 4, 7, E-cadherin, and HER2 and high expression of Vimentin, Snai 1/2, Twist 1/2, Zeb 1/2, and ALDH1, as well as stem cell characteristics. The clinical onset of claudin-low breast cancer is at menopause age, and its histological grade is higher. This subtype of breast cancer is more likely to spread to lymph nodes than other subtypes. Claudin-low breast cancer is frequently accompanied by increased invasiveness and a poor prognosis. According to a clinical retrospective analysis, claudin-low breast cancer can achieve low pathological complete remission. At present, although several therapeutic targets of claudin-low breast cancer have been identified, the effective treatment remains in basic research stages, and no animal studies or clinical trials have been designed. The origin, molecular biological characteristics, pathological characteristics, treatment, and prognosis of CLBC are extensively discussed in this article. This will contribute to a comprehensive understanding of CLBC and serve as the foundation for the individualization of breast cancer treatment. Hierarchical cluster analysis of the genes that vary more between tumors than between repeated samples of the same tumor has revealed the existence of four major breast cancer intrinsic subtypes , as well as a normal breast-like group , 2. In 22Clostridium perfringens enterotoxin (CPE)  , 12–14. in (CPE) –17. The in (CPE) . The C-tin (CPE) ), Pals1-in (CPE)  through in (CPE) –23. ZO-1in (CPE) , 25. Whein (CPE) , 27. TheWhether different claudin proteins copolymerize to form tight junction chains as heteropolymers, and whether claudin interacts in a homogeneous manner, between two molecules of the same claudin member, or between two different claudin members remains unclear. According to a previous study, different claudin members can interact within and between tight junction strands, but these combinations were restricted to specific combinations of isoforms .Claudin is widely expressed in many different tissues. The majority of claudin expression occurs in barrier-forming epithelial cells and endothelial cells. Not all claudins are expressed in all tissues at the same time, and these differences in how claudins are expressed control the functions of cells, especially paracellular barrier functions. Claudins play a key role in regulating the paracellular permeability selectivity. The overexpression of Claudin affects the resistance and permeability of epithelial cells to different ions, and ECL1 plays an important role in the selectivity of charge , 30. Var3CLBC was originally defined by a gene expression signature, which represents the low expression of claudin and cell-cell adhesion-related genes, including claudin 3, 4, and 7, and the high expression of EMT-related genes and breast cancer stem cell-related genes . Definin4ZEB1, which inhibits the activation of the E-cadherin (CDH-1) gene, activates the ZEB1 gene promoter, and promotes the transformation of mammary epithelial cells to CLBC  –55. Prev to CLBC . There i to CLBC –65. In r to CLBC . Other s to CLBC . However to CLBC , CLBC shAccording to previous studies, claudin-low breast cancer has always been considered to fall into the category of triple-negative breast cancer. According to Pommier et al. , claudin5Claudin is highly expressed in normal tissues. Although the expression of Claudin varies across tumors, it is decreased in many tumors, indicating that Claudin can inhibit the function of tumors. The increased expression of Claudin significantly inhibits the proliferation of tumor cells and promotes EMT, migration and invasion of tumors. Low expression of claudins is associated with advanced disease, metastasis, and a poor prognosis . At presApart from causing cancer in the above three aspects, Claudin also inhibits EMT. EMT is associated with tumorigenesis, progression and fibrosis . TransfoTumor metastasis is an important step in the process of tumor progression. The interaction between cancer cells and endothelial cells is the key for the distant metastasis of tumor cells. In the process of tumor metastasis, tightly connected Claudin participates in the defense of tumor cells, and tightly connected Claudin is the barrier of paracellular channels for epithelial and endothelial cell material exchange . ResearcELK3 mRNA  , 115. So6.2Several studies have shown that RAS/MAPK kinases are highly activated in breast cancer , 68, 10877.1At present, there is still controversy about the age of CLBC. Some scholars have found that patients with CLBC are younger than patients with other subtypes , 118. Ho7.2The research results of Sabatier et al.  and Xu e7.3Prat et al.  found thand Sabatier et al.  found th7.4hi/CD24-/low , and it also has low expression of cytokeratin , HER-2 and luminal-related markers , 115. ThD24-/low  and highD24-/low . RecentlD24-/low –125.Some studies have found that CLBC is heterogeneous, as is its immunohistochemical expression. Fougner et al.  believed8Prat et al.  analyzedFougner et al.  conducteTBL1 gene is related to poor prognosis and an increased metastasis rate in breast cancer patients, indicating that the expression level of TBL1 can be used as a prognostic marker (Several studies have found that many immunohistochemical markers are helpful in evaluating the prognosis of CLBC. TBL1 is necessary for the mesenchymal phenotype of transformed breast epithelial cell lines and claudin-low subtype breast cancer cell lines. The high expression of the c marker . Accordic marker , a highe9At present, research on CLBC treatment is still in the basic research stage, and most of the studies have not yet entered the animal experiment and clinical trial stages. At present, studies on the pathological complete remission of claudin-low breast cancer are based on the follow-up of the original cases. Prat et al.  followedImmunotherapy of CLBC. Fougner et al.  pointed Several novel targets have been proposed for the treatment of CLBC. Chang et al.  found th10In summary, CLBC is derived from breast cancer stem cells, luminal precursor cells, and luminal mature cells. The process of transforming to CLBC is related to EMT. We summarized the clinicopathological features of CLBC (CP and AX wrote and edited the paper, they have made the same contribution to this work and share the identity of the first author. XM, YZ and YY generated the tables and figure. CC and CW revised the paper. All authors contributed to the article and approved the submitted version.


Rates of early-onset colorectal cancer (eoCRC), defined as <50 years of age at diagnosis, have increased. Nearly one-quarter of cases of eoCRC may be associated with a germline pathogenic variant (PGV) resulting in a hereditary cancer syndrome. In the present study, we reviewed patients with a history of colorectal cancer who were referred to medical genetics at our institution to better understand the prevalence and spectrum of PGVs in both patients with eoCRC as well as with average-onset CRC (aoCRC). We found that approximately one in four patients with eoCRC had a PGV, which included 8.3% with Lynch syndrome. In patients with aoCRC, similar rates of detection of PGVs were seen. In both groups, approximately one-third of patients referred to medical genetics did not undergo genetic testing. This study reinforces the importance for patients with CRC to undergo genetic testing, especially those with eoCRC.MUTYH (4.8%), CHEK2 (3.6%), APC, BMPR1A, and TP53 (1.3% each). Among those with aoCRC, 109 patients (61%) completed genetic testing, among which 88% had either a dMMR tumor, personal history of an additional LS malignancy, or family history of LS malignancy, with PGVs detected in 23% of patients. This study reinforces the importance for all patients with CRC, especially those with eoCRC, to undergo germline testing.Over the past 20 years, rates of early-onset colorectal cancer (eoCRC), defined as <50 years of age at diagnosis, have increased, with 16–25% associated with a pathogenic germline variant (PGV) resulting in a hereditary cancer syndrome. In the present study, we sought to further characterize PGVs observed in patients with eoCRC. We conducted a retrospective analysis of patients with a history of CRC referred for genetic counseling at Mayo Clinic Rochester between April 2019 and April 2022. Three hundred and three CRC patients were referred to medical genetics, including 124 with a history of eoCRC. Only 84 patients (68%) with eoCRC referred for genetic counseling completed genetic testing, with an average of 48 genes evaluated. PGVs were identified in 27.4% with eoCRC, including 8.3% with Lynch syndrome (LS). Other detected PGVs known to increase the risk of CRC included  Colorectal cancer (CRC) is the third most common malignancy globally and is the second most common cause of cancer-related death . Over thMUTYH-associated polyposis, Peutz–Jeghers syndrome, or juvenile polyposis syndrome. Among patients with eoCRC, pathogenic germline variants (PGVs) have been reported in as many as 16–25% of cases, with Lynch syndrome accounting for up to half of these [One-third of cases of colorectal cancer have been associated with increased familial risk, with 2–10% attributed to a defined genetic syndrome ,13,14. Gof these ,16,17,18of these .At present, we are beginning to understand the spectrum of PGVs observed in eoCRC, with a significant range reported in the frequency and spectrum of germline mutation. In the present study, we evaluate patients with a history of CRC referred to medical genetics at a tertiary medical center and report individual patient data with regard to PGVs.We conducted an analysis of patients referred to Medical Genetics at Mayo Clinic Rochester between 27 April 2019 and 26 April 2022. Patients were included who had a history of CRC and were further subcategorized based on age at the time of diagnosis, <50 (eoCRC) or ≥50 ). We reviewed medical records and documented patient demographics, personal or family history of malignancy, location and stage of the tumor, tumor molecular characteristics, whether or not the patient underwent germline testing, and if so, the number of genes evaluated and results of this testing.This study was reviewed and approved (exempt) by the Mayo Clinic Institutional Review Board. Three hundred three patients with a history of CRC were referred to Medical Genetics at Mayo Clinic Rochester between 27 April 2019 and 26 April 2022. This included 124 patients with eoCRC and 179 with aoCRC. Among these, 193 patients elected to pursue germline genetic screening with results available, including 84 with eoCRC and 179 with aoCRC . Among those with eoCRC, the median age was 42 years, 59% were male, and 86% were white, non-Hispanic, whereas those with aoCRC had a median age of 62 years, 49% were male, and 93% were white, non-Hispanic . Sixty pBRAF mutations were more frequently seen in the aoCRC group (13% vs. 2%), and KRAS mutations were comparable between groups, with 22% and 19% seen in the eoCRC and aoCRC groups, respectively . The breakdown of which pathogenic mutation was observed based on MMR protein deficiency, considering BRAF V600E and MLH1 promoter hypermethylation status, is detailed in Of 84 patients with eoCRC who underwent germline testing with results available, with an average of 47.8  genes evaluated, 23 (27.4%) had an identifiable PGV, whereas 109 patients with aoCRC underwent germline testing, with an average of 48.9  genes evaluated, with 25 (23%) having a germline PGV . Among tIn the aoCRC group, 96 of 109 patients who underwent germline testing had either a tumor with dMMR, personal history of an additional Lynch syndrome malignancy, or a family history of a Lynch syndrome malignancy. All 25 PGVs identified were found in this cohort with risk factors. MUTYH (4.8%), CHEK2 (3.6%), APC, BMPR1A, and TP53 (1.3% each).In the present study, we report PGVs in 27.4% of patients with eoCRC (29.8% if including likely pathogenic germline mutations), which is comparable to rates previously reported, although on the higher end of this range ,16,17,18MUTYH, which was seen in 2.4% and 1.8% of those with eoCRC and aoCRC collectively and is associated with MUTYH-associated polyposis, which confers a 70% risk of developing CRC by age 70 [MUTYH mutation, initiating earlier CRC screening and depending on polyp burden, considering prophylactic colectomy [Detection of these PGVs can have significant implications for the patient and their family members. The most frequently observed PGVs among patients in this study were collectively found in Lynch syndrome genes. A diagnosis of Lynch syndrome conveys an increased risk of other malignancies, such as endometrial and ovarian cancer, and in those diagnosed with CRC, the decision to pursue prophylactic hysterectomy with salpingo-oophorectomy may be made at the time of surgical intervention for CRC . Additioy age 70 . NCCN guolectomy . As suchATM, BRCA1, PALB2, SDHA, and MITF. A similar phenomenon was reported by Pearlman et al., who found that 18.2% of eoCRC patients had a PGV not typically associated with CRC, including ATM, CHEK2 (classified as a less likely cause of CRC in this study), BRCA1, CDKN2A, and PALB2 [BRCA1, found in one patient with eoCRC, confers an increased risk of breast, ovarian, prostate, and pancreatic cancer. As such, a patient with a BRCA1 mutation undergoing surgical management of colorectal cancer may opt to pursue prophylactic hysterectomy with salpingo-oophorectomy at the same time considering this mutation. With regards to additional cancer screening for the patient, and other family members found to harbor this PGV, NCCN guidelines recommend more intensive screening, including initiation of annual breast MRI beginning at age 25 [In addition to genes increasing the risk of CRC, we also report PGVs not typically associated with increased risk of CRC in 7.1% of eoCRC patients, including nd PALB2 . The sigt age 25 . TherefoWhen comparing rates of PGVs in eoCRC to those observed in aoCRC, we see comparable frequency, with similar distribution of impacted genes. However, direct comparison is challenging as the aoCRC cohort in this study is a highly selected group who was referred to medical genetics for a specific reason, whether it be due to the dMMR status of their tumor, personal history of an additional malignancy , or a strong family history of malignancy. When comparing to other studies looking at rates of PGVs in patients with CRC irrespective of age at diagnosis, rates have been lower, ranging from 10–15%, which further demonstrates the selected nature of the aoCRC population in this study ,22.Current NCCN guidelines recommend multigene panel testing in patients with CRC who are diagnosed at age <50, have a tumor with dMMR, have a history of synchronous or metachronous Lynch syndrome malignancies, have a first- or second-degree relative with a Lynch syndrome malignancy diagnosed at age <50, have two or more first- or second-degree relatives with a Lynch syndrome malignancy diagnosed at any age, or have a PREMM5 score greater than 2.5% . These gHowever, whether or not the additional PGVs detected by screening all patients beyond these guidelines may impact patient management or improve survival remains to be determined. In our study, only 69% of patients with eoCRC referred to medical genetics ultimately underwent some form of germline testing, while 61% with aoCRC completed testing. Of those with aoCRC who underwent genetic testing, 88% had a dMMR tumor, personal history of an additional Lynch syndrome malignancy, or some family history of a prior Lynch syndrome malignancy. This group with high-risk features captured all PGVs among individuals with aoCRC included in this study. At this time, further study is needed to evaluate the utility of expanded germline testing, and the implications for patient outcomes.When looking more closely at patients with dMMR by IHC, loss of MLH1/PMS2 in the setting of a BRAF V600E mutation or MLH1 promoter hypermethylation, suggestive of a sporadic mutation, was more frequently seen in patients with aoCRC. Among all patients with CRC who had a loss of MLH1/PMS2 with BRAF V600E or MLH1 promoter hypermethylation, 2 of 16 were ultimately found to have a pathogenic germline mutation, but neither of these patients had Lynch syndrome, with one having an increased risk allele for prostate cancer. It is of interest that among patients with loss of other MMR proteins, 66% were found to have Lynch syndrome, which demonstrates the importance of germline analysis in this group.A limitation of this study is selection bias as patients with greater risk factors for a hereditary cancer syndrome may be more likely to pursue germline testing. This is certainly true for patients with aoCRC, and to a smaller extent, patients with eoCRC. In spite of this, this institutional study contributes to our current understanding of rates of PGVs in early-onset colorectal cancer, as well as the spectrum of mutations that are observed.In recent history, rates of eoCRC have been increasing and will make up a significant portion of new CRC diagnoses in the next ten years. Current NCCN guidelines recommend germline testing in all patients with eoCRC, and PGVs may be detected in as many as 25% of these patients. However, despite these recommendations, in the current study, nearly 1/3 of patients with eoCRC referred to genetic counseling ultimately did not pursue genetic testing. As demonstrated, the PGVs detected among this cohort can have significant implications for management for the patient, as well as their family members, and as such, demonstrates a need to increase adherence to screening guidelines. This study demonstrates the importance for patients with CRC to undergo genetic testing, especially those with eoCRC, as well as the need for further research evaluating reasons why individuals with CRC decline to pursue genetic testing.


Hyperammonemia is characterized by the excessive accumulation of ammonia in the body as a result of the loss of liver detoxification, leading to the development of hepatic encephalopathy (HE). These metabolic alterations carry cognitive and motor deficits and cause neuronal damage, with no effective treatment at present. In this study, we aimed to evaluate the effect of two subacute oral administrations of flaxseed oil (0.26 and 0.52 mL/kg) on short- and long-term memory, visuospatial memory, locomotor activity, motor coordination, and the neuronal morphology of the prefrontal cortex (PFC) via tests on Wistar rats with hyperammonemia. The goal was to identify its role in the regulation of cerebral edema, without liver damage causing cerebral failure. In contrast with an ammonium-rich diet, flaxseed oil and normal foods did not cause cognitive impairment or motor alterations, as evidenced in the short-term and visuospatial memory tests. Furthermore, the flaxseed oil treatment maintained a regular neuronal morphology of the prefrontal cortex, which represents a neuroprotective effect. We conclude that the oral administration of flaxseed oil prevents cognitive and motor impairments as well as neuronal alterations in rats with hyperammonemia, which supports the potential use of this oil to ameliorate the changes that occur in hepatic encephalopathy. Ammonia is an intermediate metabolite for protein and amino acid synthesis, and it contributes to an acid–base balance in organisms. This metabolite has the ability to cross the blood–brain barrier, and in the brain, it participates in the transformation from glutamine to glutamate. Similarly, it becomes a product of protein metabolism degradation, which is expelled from the body in the form of urea synthesized in the liver .When a liver injury occurs, regardless of its origin, high levels of ammonia are present in the blood as well as in the central nervous system, causing different effects, such as high intracellular osmolarity, which causes edema and the loss of astrocytes, releasing inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukins 1 and 6, and interferon. In the remaining astrocytes, the inhibition of α-ketoglutarate dehydrogenase and the depletion of carbon skeletons occur, consequently paralyzing the Krebs cycle .Therefore, the decreased expression of glutamate receptors in astrocytes induces increased glutamate concentrations, leading to seizures, increased cerebral blood flow, the loss of effective cerebral autoregulation, and the development of cerebral edema and intracranial hypertension . This isThis disease comprises a clinical picture of various neuropsychological disorders, with cognitive, emotional, personality, motor activity, memory, and consciousness manifestations, and patients can even fall into a coma. It can episodically or continuously appear and is reversible in most cases . There iLinum usitatissimum seeds, and it contains various compounds including caffeic acid, p-coumaric acid, ferulic acid, secoisolarisiresinol diglucoside, and secoisolariciresinol. Additionally, this oil is abundant in unsaturated fatty acids, such as oleic, linoleic, palmitic, and stearic acids, with linoleic acid being the most prevalent, constituting nearly half of the total fatty acids. Moreover, flaxseed oil is rich in polyphenolic compounds, which contribute to its significant antioxidant potential [The current treatment for this disorder involves the removal of ammonia in the blood; hence, antibiotics and non-absorbable disaccharides are used, mainly rifaximin and lactulose . Howeverotential . Linoleiotential ,13,14,15otential ,19,20,21Flaxseed is commonly available for human consumption in three different forms: whole, ground, and oil flaxseed. Their stability, bioactive components, and pharmacokinetic parameters depend on the way they are consumed. The alpha-linolenic acid (ALA) in whole seeds withstands temperatures of up to 350 °C and maintains oxidative stability, but when the seed’s outer shell is crushed, this stability decreases. This oil should be stored in opaque glass containers, be refrigerated, and have an unrefined form to prevent oxidation and preserve its bioactive properties . FlaxseeTherefore, in this study, we aimed to evaluate the cognitive and motor effects of orally administrating flaxseed oil to model rats with hyperammonemia who ate an ammonium-containing diet to identify its role in regulating cerebral edema, without hepatic damage causing cerebral failure.We carried out experiments on animals in accordance with the guidelines of the Care and Use of Laboratory Animals  and Norma Oficial Mexicana para el Cuidado y Uso de Animales de Laboratorio . Additionally, the 3Rs principles  were taken into consideration in preclinical research .® from GNC . Prior to administration, the oil from each capsule was extracted using a removable insulin syringe needle. The capsule’s content was analyzed using mass spectrometry, and a total of 43% alpha-linolenic acid was determined per capsule.Flaxseed oil was obtained from the capsules of the dietary supplement Flax Seed Oil 1000Male Wistar rats weighing 150–200 g were used in this study. They were kept in a vivarium in the Faculty of Pharmaceutical and Biological Chemistry of the Universidad Veracruzana in translucent acrylic boxes (45 × 30 × 10 cm) with a 12 h light/dark cycle (lights on at 7:00 a.m.), with free access to water and food (4 per box).w/w ammonium acetate. For eight weeks, 28 rats had access to an ammonium-containing diet ad libitum [An ammonium-containing diet was prepared from dry rodent food mixed with 20%  libitum .n = 7 each): (A) hyperammonemia control (HaC); (B) ibuprofen 30 mg/kg/day ip (HaIbu); (C) flaxseed oil ig 0.26 mL/kg/day (HaFOD1); and (D) flaxseed oil ig 0.52 mL/kg/day (HaFOD2). An additional group was used as a control, which was administered mineral oil ig and fed a standard diet for the duration of the experiment (control group). All the treatments were administered during the final 3 weeks of the experiment. Once the treatments were completed, behavioral tests were conducted, followed by blood and brain tissue collection. These two doses of flaxseed oil were based on the Mantzioris et al. 2000 study and adjusted after analyzing the composition of alpha-linolenic acid using mass spectrophotometry with a nutritional supplement . Additionally, Rodrigo et al. (2010) justified the use of intraperitoneal ibuprofen at a dose of 30 mg/kg to reduce neuroinflammation induced by hyperammonemia in an animal model of encephalopathy without liver damage, as well as in the bile duct ligation model [On the fifth week, the 28 rats were divided into four different treatments  was conducted, and after 24 h, the number of squares crossed by the animal after 5 min was evaluated, as well as the frequency of rearing activity and the time spent grooming.To evaluate short- and long-term memory, the spontaneous object recognition test (SOR) was used, in which short-term memory was measured one hour after the end of the training phase, and long-term memory was measured 24 h later. Once exploration duration data were obtained, the percentage of time spent exploring the novel object during each test was calculated relative to the total exploration time spent focused on either of the two objects.The Barnes maze was used to evaluate visuospatial memory. Ten trials were conducted per subject in the acquisition phase, and 24 h after the trials were completed, the memory test was performed. The escape latency  was used) was determined. Likewise, the number of errors made during the acquisition phase were checked (primary errors); this refers to the instances where the subject inserted their head into incorrect holes before reaching the goal or successfully escaping.Motor coordination was evaluated using the Rotarod test. An automated 4-line Rotarod RS system  for rats was used, in which the animals were placed in a 7 cm diameter cylinder, after habituating for 3 min for two consecutive days with the apparatus turned off. On the third day, the test was performed with a speed from 4 to 40 rpm for 300 s, during which the latency until the first fall post-treatment administration was measured, with a maximum cut-off point of 600 s .After conducting the behavioral experiments, the animals were euthanized via decapitation after being anesthetized with Xylazine  (2 mg/kg). Their brains were extracted and stored in 4% formaldehyde solution in phosphate buffer until further use. They were refrigerated at 4 °C for 24 h before being transferred to phosphate buffer pH 7.4 for 1 h before sectioning. Coronal sections of 30 μm thickness were obtained using a Leica VT 1200 S vibratome  and subsequently stained using the hematoxylin-eosin technique. From each brain, 50 sections were obtained, and an average number of neurons was calculated. Neuronal counting was performed via the optical microscopy of layer III of the prefrontal cortex; these images were acquired using Leica Application Suite in conjunction  with a Leica DMi1 microscope . Then, 500 microns were measured inward, creating a rectangular area of 250 × 500 microns for analysis. The presence of degenerated cells was determined by counting neurons exhibiting pyknosis, karyorrhexis, karyolysis, cytoplasmic eosinophilia, or the loss of hematoxylin affinity, as well as dark, festooned, or edematous neurons.p ≤ 0.05. The results were plotted using GraphPad Prism 6 software (Prism Mac 6.0h). Data are presented as the mean and standard error of the mean.All data were analyzed using the R software version 4.3.1 through one-way ANOVA for independent samples. Tests that did not meet the normality criteria were analyzed using non-parametric statistics. All statistical tests were two-sided, and a post hoc Tukey test was performed when  = 2.673, p = 0.051, NS). On the other hand, the hyperammonemic group reared more  = 4.959, p = 0.003) and spent less time grooming  = 5.391, p = 0.002). = 7.144, p = 0.0003), an effect that was prevented with both doses of flaxseed oil treatment (HaFOD1 and HaFOD2) but not with the ibuprofen prototype (HaIbu). = 3.977, p = 0.01). However, in the test conducted 24 h after the familiarization phase, no significant differences in the exploration of objects were observed in any of the groups  = 0.44, p = 0.779).e groups B  = 11.552, p = 8.527 × 10−6), indicating that they spent more time locating the goal during the trials. Similarly, in the 24 h test  = 10.72, p = 1.62 × 10−5). However, the groups treated with both doses of flaxseed oil and ibuprofen showed a reduction in the time spent finding the goal during both the acquisition phase and the 24 h test  = 10.72, p = 1.62 × 10−5). For the number of errors made before reaching the goal, the hyperammonemic group performed more errors than the other groups did  = 22.31, p = 0.001).4 h test B, the hyoups did C  = 156.23, p < 0.001). The other hyperammonemic groups treated with ibuprofen and flaxseed oil suffered less neuronal damage and had more normal neurons than the hyperammonemic control group did. However, their neuron count was still not as high as that of the control group fed a standard diet  = 156.23, p < 0.001).As seen in This study aimed to investigate the potential neuroprotective and behavioral effects of flaxseed oil in a hyperammonemia model induced by an ammonia-containing diet in Wistar rats. The study demonstrated that the flaxseed oil and ibuprofen treatments improved their short-term memory, visuospatial memory, locomotor activity, and motor coordination and preserved the normal neuronal morphology of the prefrontal cortex. The study involved an eight-week ammonia-containing diet regimen, with treatments of flaxseed oil and ibuprofen administered during the final three weeks.An open field test (OFT) and a Rotarod test were utilized to evaluate locomotor activity and motor coordination. The OFT was also used to assess affective behaviors, such as the time spent grooming and rearing. Hyperammonemia leads to motor coordination impairments and anxious-like behaviors, and increased activity in animals may be due to neurological damage . During The prefrontal cortex (PFC) is involved in cognitive processing that supports executive functions such as problem solving, memory, and learning. Hepatic encephalopathy causes neuronal damage characterized by a decrease in the number of healthy neurons and the appearance of Alzheimer type II astrocytes. These characteristics are associated with cognitive and motor function impairments resulting from a cerebral edema, leading to typical symptomatic patterns . Some auThis study shows that the control group fed a standard diet exhibited a normal neuronal morphology, without alterations, and with a cell count that follows a consistent pattern. However, the subjects of the hyperammonemic control group fed an ammonium-containing diet begin to exhibit an atypical neuronal morphology as well as a tendency toward having fewer neurons per field, which could lead to the cognitive impairment observed in the subjects. The treatments with ibuprofen and both doses of flaxseed oil restored the neuronal morphology of the subjects. The potential neurological effect of flaxseed oil is associated with its high content of alpha-linolenic acid, which has been demonstrated in several studies to be neuroprotective ,37,38,39Therefore, we used the Barnes maze test (BMT) and spontaneous object recognition (SOR) test to evaluate different types of memory. The BMT is used to assess visuospatial memory, while SOR tests short- and long-term memory. In this study, the control group had a lower escape latency in both the BMT and 24 h test. In contrast, the group fed an ammonium-containing diet took more time to escape during the 10 trials and 24 h test, indicating a cognitive impairment. Interestingly, there were no differences in exploring both objects in the SOR test, suggesting a lack of discrimination between the two. This is consistent with the idea that high levels of ammonia can cause brain toxicity and cognitive damage in patients with HE and other animal models.Ibuprofen exerts an anti-inflammatory effect by crossing the BBB and inhibiting the production of cyclooxygenases 1 and 2 (COX-1 and COX-2), thus reducing the production of prostaglandins, thromboxanes, and leukotrienes. This explains its observed effect on cerebral edema. On the other hand, although the mechanism of action of flaxseed involved in the present study was not identified, it is possible to suggest that oral administration provides alpha-linolenic acid (ALA), which can undergo β-oxidation in the liver to be transformed into docosahexaenoic acid (DHA) and cross the blood–brain barrier (BBB). Moreover, ALA can also cross the BBB with the assistance of transporter proteins. In turn, DHA inhibits the production of inflammatory cytokines, thereby reducing edema.On the other hand, a diet rich in omega-3 fatty acids, particularly linolenic acid, and low in omega-6 fatty acids, such as linoleic acid, has been shown to have a neuroprotective effect ,42,43,44®). We chose flaxseed oil due to it being one of the primary sources of omega-3 . Additionally, it contained an appropriate proportion of omega-6 and omega-9, necessary for its biological effect. Our intention was not to evaluate isolated bioactive compounds but rather to evaluate the oil. Perhaps, in future research, it would be interesting to assess the behavior of isolated bioactive components to determine if their effects are reduced or enhanced.In our study, we did not measure the plasma concentration of bioactive compounds, nor that of flaxseed oil, in plasma or tissue prior to the behavioral measurements. This objective was not part of our study; however, we believe that it is a natural progression in this line of research to explore pharmacokinetic parameters and variations in the bioavailability of different sources of flaxseed oil or even omega-3 itself and relate them to the biological effect. On the other hand, the two doses of flaxseed oil were based on the Mantzioris et al. 2000 study and on the analysis of the composition of alpha-linolenic acid using mass spectrophotometry with a nutritional supplement (Flax Seed Oil 1000Flaxseed oil restores motor and memory impairments, as well as neuronal morphology alterations caused by a high-ammonium diet, possibly due to its potential anti-inflammatory properties. It improves the neuronal, cognitive, and motor characteristics of rats with hyperammonemia.


Hot spring outflow channels provide geochemical gradients that are reflected in microbial community compositions. In many hot spring outflows, there is a distinct visual demarcation as the community transitions from predominantly chemotrophs to having visible pigments from phototrophs. It has been hypothesized that this transition to phototrophy, known as the photosynthetic fringe, is a result of the pH, temperature, and/or sulfide concentration gradients in the hot spring outflows. Here, we explicitly evaluated the predictive capability of geochemistry in determining the location of the photosynthetic fringe in hot spring outflows. A total of 46 samples were taken from 12 hot spring outflows in Yellowstone National Park that spanned pH values from 1.9 to 9.0 and temperatures from 28.9 to 92.2°C. Sampling locations were selected to be equidistant in geochemical space above and below the photosynthetic fringe based on linear discriminant analysis. Although pH, temperature, and total sulfide concentrations have all previously been cited as determining factors for microbial community composition, total sulfide did not correlate with microbial community composition with statistical significance in non-metric multidimensional scaling. In contrast, pH, temperature, ammonia, dissolved organic carbon, dissolved inorganic carbon, and dissolved oxygen did correlate with the microbial community composition with statistical significance. Additionally, there was observed statistical significance between beta diversity and the relative position to the photosynthetic fringe with sites above the photosynthetic fringe being significantly different from those at or below the photosynthetic fringe according to canonical correspondence analysis. However, in combination, the geochemical parameters considered in this study only accounted for 35% of the variation in microbial community composition determined by redundancy analysis. In co-occurrence network analyses, each clique correlated with either pH and/or temperature, whereas sulfide concentrations only correlated with individual nodes. These results indicate that there is a complex interplay between geochemical variables and the position of the photosynthetic fringe that cannot be fully explained by statistical correlations with the individual geochemical variables included in this study. In the phototrophic mats below the photosynthetic fringes of basic springs (pH > 7), the microbial communities consist predominantly of bacterial phototrophs, including Cyanobacteria and filamentous anoxygenic phototrophs . The prefixation . Below toschyzon . Both euoschyzon . Thus, ioschyzon .The predominantly chemotrophic communities above the photosynthetic fringe of hot spring outflows also vary with pH . In gene13C) data as well as DIC and DOC concentrations. In the outflow of a basic spring, “Bison Pool,” also discussed in this study as hot spring “BP,” Δ13C measurements of the biofilm became more negative down the outflow, indicating a possible shift in carbon fixation strategies  and dissolved organic carbon (DOC). rategies . Additiog meadow . In the g meadow .nif) genes only at and below the photosynthetic fringe. In contrast, the expression of nif genes was observed above the photosynthetic fringe at “Mound Spring,” also discussed in this study as hot spring “MN”  to 52.6 μmolal . Due to below (chemosynthetic and photosynthetic), at (fringe), and above (chemosynthetic) the photosynthetic fringe in hot spring outflow channels, though additional samples were taken at several sites to provide additional biogeochemical context. The LDA model (Equation 1) was trained to separate samples into photosynthetic and non-photosynthetic classes based on 20 variables measured across 56 samples collected from twenty-nine geochemically diverse hot springs in previous years. The 20 variables selected to construct the LDA model were chosen based on perceived biological relevance, specifically, temperature, pH, conductivity, DIC, DOC, DO, Fe(II), sulfide, phosphate, total ammonia, and total dissolved Mg, Co, Ni, Cu, Zn, As, Mo, Cd, W, and Pb. Fe(II) was used in place of total Fe because Fe(II) could be measured in the field spectrophotometrically. Additionally, representative variables were chosen per element, for example, total ammonia is representative for nitrogen species. Samples above the photosynthetic fringe tend to have greater negative LDA scores, while samples below tend to have greater positive scores. Photosynthetic fringe positions were determined visually when possible and by LDA when not discernable by eye. When testing the LDA model on 381 previously collected samples, where photosynthesis had been identified by the visual presence of photosynthetic pigments, an LDA score of 0.13 predicted photosynthetic and non-photosynthetic samples with the fewest number of false positives and negatives. In outflow channels where the fringe was not apparent, the location of the fringe was estimated by choosing a location where the LDA score was close to or equal to 0.13 based on temperature, pH, and conductivity measured in the field combined with historical data for the remaining 17 variables in the LDA model. In the outflow of CF and MO, the photosynthetic fringe was apparent but occurred at different temperatures throughout the hot spring outflow, hence multiple samples were taken throughout the outflows to account for these variations. Sampling locations were chosen such that they were chemically equidistant from the fringe as estimated by the LDA model. In other words, sampling was carried out such that the difference in LDA scores between the at and below samples was equal to the difference between above and at scores. The LDA model was trained using the lda function in the MASS package in R (RRID:SCR_019125) (Sampling locations  were det_019125) .Equation 1.Sigma symbols indicate that the variable includes measured values of total solute concentrations and includes all protonation states and complexes.Temperature, pH, and conductivity were measured in the field as previously described . TemperaFiltered  water samples for laboratory analyses were collected and stored according to previously described procedures . Samples–, Cl–, SO4–2, Br–, NO3–) and cations  were determined on separate Dionex DX-600 4 mm ion chromatography systems using suppressed-conductivity detection as described elsewhere (RRID:SCR_016874) gradient applied over 32 min to 55 mM hydroxide, after which the concentration was kept constant at 55 mM for 7 min, reduced back to 5 mM hydroxide over 1 min, and then the column was re-equilibrated at 5 mM hydroxide for 10 min before the next sample injection. The flow rate was held constant at 1 ml/min. Cations were separated isocratically using 19 mM methanesulfonic acid on CG-/CS-16 columns at 0.5 ml/min over 58 min. Suppressors were operated in external water mode and suppressor currents were 137 and 50 mA for anions and cations, respectively. Calibration curves were constructed from a series of dilutions of mixed-ion standards  and accuracy was verified daily by analysis of an independent mixed-ion standard (Thermo Scientific).Anions (Flsewhere . Samples2 was generated via addition of phosphoric acid (DIC) or sodium persulfate (DOC) and the ion chromatogram for the molecular ion (44 m/z) was used for quantification relative to calibration curves prepared with sodium bicarbonate (DIC) or glycine (DOC) standards. Three sample loops with volumes of 1 ml , 5 ml , and 25 ml  were employed to capture the range of carbon concentrations across the sample set.Analyses of DIC and DOC were performed with a OI Wet Oxidation TOC analyzer coupled to a Thermo Delta Plus Advantage mass spectrometer as previously described . BrieflyHot spring outflow sediment samples were collected and preserved for biological analyses . Samples were collected using a flame-sterilized spatula into a 1.8 ml cryovial. Once samples were collected, they were transferred into a container of dry ice and frozen until they could be stored at −80°C at ASU.RRID:SCR_020134) with the Earth Microbiome Project primers 505F and 806R (1 (RRID:SCR_004891) under BioProject ID PRJNA938133.For DNA extraction, biological samples were homogenized, and DNA was extracted using a ZymoBIOMICS DNA Miniprep Kit , binding capacity 25 μg, as previously described . A NanoDand 806R . The 16S 806R (1 . All rawRRID:SCR_014583) was used to quality filter the 16S rRNA amplicon sequences (RRID:SCR_021258) pipeline to produce amplicon sequence variants (ASVs) and were denoised using the DADA2 plug-in (RRID:SCR_006423) was used for the taxonomic classification of ASVs (RRID:SCR_011950) and Bray-Curtis Dissimilarity values  function in the VEGAN package. To determine the significance of separating sites as above, at, or below the photosynthetic fringe, analysis was performed using the canonical correlation analysis (CCA) function from the VEGAN package and an ANOVA test was performed on each axis using the R stats package. For the co-occurrence network analysis, ASVs were filtered to only include ASVs that had more than 20 reads across all samples and occurrences across more than 3 sample sites. The remaining ASVs were used to construct a co-occurrence network using R’s igraph package (v. 1.2.11) (RRID:SCR_021238), in which each node is an ASV, and each edge represents a Spearman’s correlation coefficient greater than 0.7 between the two nodes (RRID:SCR_004293) based on their Spearman correlation with the selected geochemical variables (FASTQC (v. 0.11.9)  to determine the locations above and below the photosynthetic fringe such that sampling would be equidistant from the fringe in multivariate geochemical space. The photosynthetic fringe can often be visually confirmed as seen in example images of a sampled basic  and an aIn total, 46 samples were taken with the intention of spanning the temperature, pH, and sulfide ranges provided by YNP hot springs . Of the The relative abundance of taxonomic classes present in each of the 46 samples was determined by 16S rRNA gene sequencing to investigate the microbial community composition and diversity along each outflow and among the hot springs sampled . In eachLeptococcus and Chloroflexus, respectively, occurred with higher relative abundances in the circumneutral to basic hot spring outflows. Both Cyanobacteria and Chloroflexi occurred in nearly all hot spring outflows sampled, except, neither Cyanobacteria nor Chloroflexi occurred in the outflow of GL  or MO , nor did Chloroflexi occur in the outflow of FI . Furthermore, neither Cyanobacteria nor Chloroflexi surpassed a relative abundance of 0.1% in the outflow of any of the acidic hot springs sampled. Chloroflexi reached relative abundances above 1% only at pH values greater than 7, while Cyanobacteria reached a relative abundance above 1% only at pH values greater than 8. In basic conditions, Chloroflexi and Cyanobacteria made up to 30.7 and 25.6% of the microbial community, respectively, at their highest relative abundances. Chloroflexi occurred above, at, and below the visually determined photosynthetic fringe. However, Chloroflexi reached higher relative abundances at (0.9–2.4%) and below (0.0–22.6%) the photosynthetic fringe compared to above (0.0–3.4%). Cyanobacteria were also identified above the visually detected photosynthetic fringe but did not surpass a relative abundance of 1.8% except at outflow sample BP0.5, where the relative abundance was 6.2%. In contrast, at and below the photosynthetic fringe, the relative abundances for Cyanobacteria made up to 25.6% of the microbial community. ASVs associated with putative photoheterotrophs were also present, including those in the taxonomic classes of Alphaproteobacteria and Acidobacteriia, such as Acidiphilium, Acidisphaera, and Chloracidobacterium, which, when combined, only surpass a relative abundance of 1% in a single sample, MU3, where they make up 4.0% of the community.Amplicon sequence variants associated with photoautotrophs, including those in the phyla Cyanobacteria and Chloroflexi, such as Samples at or below the photosynthetic fringe indicate an apparent cut-off for photosynthesis at ∼73°C for basic to circum neutral pH samples . The temIn addition to ASVs associated with photosynthetic taxa occurring in the circumneutral to basic hot spring outflows, ASVs associated with non-phototrophs in the taxonomic classes of Acetothermia, Kapabacteria, Fervidibacteria, Hydrothermae, Anaerolineae, and the phylum Armatimonadota were also present. Although Cyanobacteria and Chloroflexia were abundant within the outflow of basic hot springs, ASVs associated with the non-photosynthetic phylum Armatimonadota were also prevalent in these samples, occurring with an average relative abundance of 13.5% when present. Armatimonadota were present in every sample above a pH of 7, except for sample site 0.5 from BP. As for the ASVs associated with the taxonomic classes Acetothermia, Kapabacteria, Fervidibacteria, Hydrothermae, and Anaerolineae, they reached relative abundances above 1% only above a pH of 7 but none were as consistently predominant as ASVs associated with the phylum Armatimonadota.The ASVs abundant in the lower pH sites (pH < 7) include ASVs from the following taxonomic classes: Gammaproteobacteria, Alphaproteobacteria, Desulfurellia, Actinobacteria, Acidimicrobiia, and Thermoplasmata. Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria were present in at least one sample from each hot spring outflow but reached relative abundances above 1% only at acidic sites. Acidimicrobiia were present in all hot springs sampled except for in the outflow of MN  and only reached a relative abundance over 10% at pH values below 3. Gammaproteobacteria had a high relative abundance of 61.8% at the photosynthetic fringe of hot spring CF where the temperature was 28.8°C and the pH was 3.7. Thermoplasmata and Desulfurellia were restricted to circumneutral and acidic sites, only occurring below a pH of 8.0 and 6.5, respectively, with Desulfurellia only making up to 5.3% of any microbial community composition, while Thermoplasmata reached up to 34.5% of the relative abundance of the microbial community composition below the photosynthetic fringe of CF.Additionally, there were ASVs associated with three taxonomic classes that were restricted to circumneutral conditions, including Ktedonobacteria, Bacteroidia, and Bathyarchaea. Ktedonobacteria only occurred with a relative abundance higher than 1% at the photosynthetic fringe of MU, where they made up 32.2% of the community, which is at a pH of 5.7. Bacteroidia occurred across pH, but only made up more than 1% of the community in the outflows of MU, FI, and EM, which range in pH from 3.9 to 8.0. Below the photosynthetic fringe of FI, Bacteroidia made up 39.0% of the microbial community. Bathyarchaea were present in small relative abundances (<0.1%) in the outflows of RN and CF but occurred with relative abundances of 13.6 and 12.6% at the photosynthetic fringes of FI and EM, respectively. The photosynthetic fringe of FI occurred at a pH of 5.8 while the photosynthetic fringe of EM occurred at a pH of 7.8.Overall, there were trends in microbial community composition across pH as well as down the outflows of the 12 sampled hot springs. In the lower pH sites, ASVs associated with taxonomic classes such as Thermoplasmata and Gammaproteobacteria dominated. In basic sites, ASVs associated with the phylum Armatimonadota dominated, as well as ASVs associated with the potentially photosynthetic phyla, Cyanobacteria and Chloroflexi. The relative abundance of potential bacterial photosynthetic taxa increased in relative abundance with increasing pH and increased down hot spring outflows. In contrast, the relative abundance of the taxonomic class Hydrothermae decreased down hot spring outflows. At all hot springs and outflows, ASVs for the taxonomic classes Aquificae and Deinococci were present and made up a considerable portion  of the microbial community composition.p > 0.05) except for sulfide. Along NMDS2, the distribution of sites can be differentiated based on position relative to the photosynthetic fringe. However, only sites labeled as being above the photosynthetic fringe can be differentiated from the sample sites at or below the photosynthetic fringe with statistical significance according to CCA analysis  analysis was used to interrogate the influence of geochemistry on microbial community composition across the photosynthetic fringe . The 16Sanalysis . The geoWhile NMDS analyses provide insight into overall microbial community composition in relation to geochemical parameters, co-occurrence network analyses provide insight at the level of individual ASVs and their correlation(s) with geochemical parameters . In a coThe co-occurrence network consisted of ten cliques with no interconnections (modular cliques), a central cluster consisting of six interconnected cliques, and a separate cluster of two cliques. This topology suggests there are groups of ASVs that uniquely co-occur (modular cliques) and groups of ASVs that co-occur predominantly with each other (main cluster cliques). Some members of the main cluster cliques co-occur with ASVs outside the clique as well. The statistical association of the cliques with geochemical parameters of interest was conducted by Spearman correlation. From these combined analyses we can observe how cliques and individual ASVs co-varied with pH, temperature, total ammonia, DIC, DOC, nitrate, and sulfide concentrations .Within the main cluster of six cliques  all ASVs correlated positively with an increase in pH . The remLeptococcus, Roseiflexus, and Chloroflexus, as well as non-phototrophs from the taxonomic classes Aquificae, Anaerolineae, Deinococci, Acetothermiia, and the phylum Armatimonadota among others listed in Thermocrinis and Thermus, respectively. Although all 6 cliques within the main cluster are correlated positively with an increase in pH, only cliques 2, 4, and 9, which include ASVs associated with the taxa Thermocrinis, Deinococci, Leptococcus, Acetothermia, and Armatimonadota, are positively correlated with temperature. Clique 5 is correlated slightly negatively with temperature and consists of the taxonomic classes Anaerolineae, Acetothermia, and the phylum Armatimonadota. Additionally, cliques 2, 4, and 9 are all correlated negatively with total ammonia, whereas the nodes in clique 5 are mixed between positive and negative correlations with total ammonia. The remaining cliques in the cluster (8 and 13) are not correlated with total ammonia. In contrast, all 6 cliques within the cluster are correlated negatively with DOC, although cliques 2 and 9 have a stronger negative correlation with DOC than cliques 4, 5, 8, or 13.The main cluster cliques consist of the phototrophs Meiothermus, and Aquificae is represented by Hydrogenobaculum. Cliques 7 and 11 are correlated positively with total ammonia and DOC concentrations but are correlated negatively with pH and temperature. The only major differences in trends for cliques 7 and 11 are displayed in Outside of the main cluster cliques, there are five cliques  that are correlated negatively with pH. Cliques 7 and 11 are interconnected and tend to follow similar trends. Cliques 7 and 11 include ASVs associated with the taxonomic classes Acidimicrobiia, Thermoplasmata, Deinococci, and Aquificae. In this case, Deinococci is represented by Meiothermus, Acidimicrobiia, Gammaproteobacteria, Alphaproteobacteria, and Thermoprotei, are correlated positively with total ammonia and DOC concentrations. However, cliques 3 and 12 are correlated negatively with pH but not temperature, whereas clique 15 is negatively correlated with both temperature and pH.The unconnected cliques, 3, 12, and 15, are also correlated negatively with pH. Cliques 3, 12, and 15, which contain the taxa Ignisphaera, a Thermoprotei, is positively correlated with both temperature and pH but negatively correlates with both total ammonia and DOC. Clique 17 contains Nitrososphaeria and Thermoprotei and is correlated positively with temperature, but has no strong correlation with total ammonia or DOC.In contrast to the previously mentioned cliques, cliques 17 and 18 are correlated positively with an increase in pH and are outside of the central cluster. Clique 18, which only contains two taxa, Fervidicoccaceae and Thermoflavifilum, whereas clique 10 contains the taxa Geoarchaeales and Corynebacterium. Clique 14 is unique in that it is correlated negatively with temperature but has a slight positive correlation with total ammonia. The taxa in clique 14 include Meiothermus, Betaproteobacteria, and Gammaproteobacteria. Clique 1, which consists of Mycobacterium and Thiomonas, is correlated positively with temperature and correlated negatively with total ammonia, but is not strongly correlated with temperature or DOC.The remaining cliques, 1, 6, 10, 14, and 16, lack strong correlations with pH in either direction. Cliques 6, 10, and 16 are only correlated with temperature, cliques 6 and 16 correlated negatively with temperature, and clique 10 correlated positively with temperature. Clique 6 is correlated negatively with temperature and total ammonia concentration but is correlated positively with DOC. Besides temperature, cliques 10 and 16 cannot be differentiated based on pH, total ammonia, or DOC. However, clique 16 is correlated negatively with sulfide and is positively correlated with nitrate . There aMeiothermus, from the class Deinococci, and Armatimonadota, both of which are found across all hot springs sampled.Overall, we find internal consistencies between the pH of samples where ASVs are abundant and the correlation with pH in the co-occurrence networks. For example, taxa associated with higher pH samples in p < 0.05) through an ANOVA test of the CCA axes  and were determined to contribute 7.33 and 4.85%, respectively, to variations in the microbial community compositions according to RDA, in agreement with previous studies   . Additio = 0.30) , in whic2 gas as temperature decreases and the increasing extent to which the reduced hydrothermal fluids have equilibrated with the atmosphere. In sulfidic systems, DO is quickly consumed in abiotic oxidation reactions, including the oxidation of reduced sulfur compounds  within microbial communities, as well as overall microbial community composition down hot spring outflows. Ammonia and nitrate contribute 3.78 and 2.55% to the variation of overall microbial community . Further springs . However springs . Thus, t2, which increases the pH for already circumneutral to basic hot springs  can be found in the article/ES, GB, and KF conceived the study and supervised the sampling. AH, FD, and GG carried out the DNA extractions, library prep, and sequencing efforts. KW, AH, and ET-R performed the sequence processing and bioinformatic analyses. KF performed the processing of water samples and ion chromatography (IC). KW and ET-R conducted the in-depth statistical analyses of geochemical and microbiomic data, including NMDS, and the co-occurrence network analysis. KW and ET-R wrote the manuscript with input from all authors. All authors contributed to the article and approved the submitted version.