COL5A1 gene, rs2721051 near the FOXO1 gene, rs1324183 near the MPDZ gene) are also actual for a Russian cohort of patients. Additionally, we investigated the COL5A1 promoter sequence for single-nucleotide variants (SNVs) in a subgroup of keratoconus patients with at least one rs1536482 minor allele (rs1536482+) to assess the role of these SNVs in keratoconus susceptibility associated with rs1536482.Keratoconus is a chronic degenerative disorder of the cornea characterized by thinning and cone-shaped protrusions. Although genetic factors play a key role in keratoconus development, the etiology is still under investigation. The occurrence of single-nucleotide polymorphisms (SNPs) associated with keratoconus in Russian patients is poorly studied. The purpose of this study was to validate whether three reported keratoconus-associated SNPs . We performed PCR targeting regions flanking SNVs and the p-value < 0.05). The rare variants rs1043208782 and rs569248712 were found in the COL5A1 promoter in two out of 94 rs1536482+ keratoconus patients.The minor allele frequency was significantly different between the keratoconus and control cohorts (main and combined) for rs1536482, rs2721051, and rs1324183 (COL5A1 promoter do not play a major role in keratoconus susceptibility associated with rs1536482.rs1536482, rs2721051, and rs1324183 were associated with keratoconus in a Russian cohort. SNVs in the  Keratoconus is the most common primary ectatic disorder associated with thinning, stretching, and conical protrusion of the central part of the cornea. The progression of this disease can lead to severe visual impairment due to corneal irregularities and scarring. The disease most often manifests in adolescence and progresses within 10–15 years . The preCOL5A1 gene, near the FOXO1 gene, and near the MPDZ gene. Initially, all three loci were discovered to be associated with central corneal thickness in GWASs [FOXO1 and MPDZ were also associated with corneal hysteresis and corneal resistance factors [Understanding the genetic basis of keratoconus can facilitate its early diagnosis . The genin GWASs –17. Thenin GWASs , 13, 14. factors , 19.Keratoconus susceptibility loci, even those reported previously, are poorly studied in Russian patients with keratoconus. Patients with keratoconus from Russia were previously genotyped for rs1536482 and rs7044529 in our pilot study on a small sample .COL5A1 gene, rs2721051 near the FOXO1 gene, rs1324183 near the MPDZ gene) with keratoconus in the largest sample of patients from Russia. Additionally, we investigated rare variants in the COL5A1 promoter of keratoconus patients with at least one rs1536482 minor allele.This study aimed to investigate the replication of three SNPs , were excluded. The patients were all of the European descent.Clinically affected patients with keratoconus were recruited at the S. Fyodorov Eye Microsurgery Complex Federal State Institution and the Ufa Eye Research Institute. All patients underwent clinical and functional examination, visual acuity testing, refractometry, ophthalmometry, biomicroscopy, and ophthalmoscopy. During slit-lamp examination, surface asphericity and the presence of thinning, Vogt’s striae, Fleischer’s rings, Descemet’s membrane folds, and stromal scarring were evaluated. Keratoconus patient corneas were also assessed using optical coherence tomography Visante OCT  and corneal topography TMS-4 . Characteristic keratoconus patterns of corneal topography images ), increased surface asymmetry index and surface regularity index values (> 1) along with a central thickness of less than 480 nm, and an inability to correct visual acuity to 1.0 (20/20) were critical for the differential diagnosis of early disease. The keratoconus stage was determined according to the Amsler-Krumeich classification . PatientCOL5A1 promoter.Patients diagnosed with keratoconus were also asked if they would agree to invite first-degree relatives. If they agreed, then their relatives were invited and consulted about participation in the study. After obtaining written informed consent, the relatives were examined in the same way as the keratoconus patients. Participation also meant that blood collection was allowed. For 50 of the patients with keratoconus, the material was also obtained from relatives. The relatives’ DNA was used only for the cosegregation analysis of variants found in the Participants in the main control group (n = 205) were evaluated at the S. Fyodorov Eye Microsurgery Complex Federal State Institution and the Ufa Eye Research Institute. Medical history collection and a thorough ophthalmic examination were carried out for all potential participants. Patients with keratoconus, glaucoma, medium- to high-degree myopia, or retinal or corneal dystrophy were not included. Acceptable ocular diseases included mild myopia, pseudoexfoliation syndrome, and senile (age-related) cataracts. No subjects with a medical history of early-onset cataracts were included, as early-onset anterior polar cataracts are associated with keratoconus . ParticiA control group of 503 participants, described by Levchenko A. et al., was included as an additional control group . BrieflyVenous blood (4–6 mL) was collected from each participant in Vacutainer tubes with EDTA . The samples were stored at − 20 °C until genetic study. DNA was isolated from blood samples with the Gentra Puregene Blood Kit  according to the manufacturer’s protocol. DNA was resuspended in low-TE buffer at a final concentration of 10 ng/μl.SNV genotyping was carried out by Sanger sequencing. The primer sequences for rs1324183 and rs2721051 were obtained from an article by Liskova P. et al.  [To evaluate st exon) . This rest exon) . PrimersFor the amplification of target sequences, we used the Gene Pak PCR MasterMix Core Kit  under the following conditions: reaction volume, 20 μl; DNA input, 50 ng; and final concentration for each primer, 0.3 nM. Sanger sequencing was carried out according to the manufacturer’s protocol with the BigDye Terminator v3.1 Kit (Thermo Fisher Scientific) on an ABI Prism 3730XL Genetic Analyzer .An additional control group was genotyped by Levchenko A. et al. using the iScan System and the Infinium Global Screening Array-24 v1.0 BeadChip  . We convGenotypes were initially presented in the SNP array data for only rs1536482 and rs2721051, which could thus be directly analyzed by PLINK. The MAF and minor homozygote frequency (MHF) for rs1324183 SNV were estimated after the imputation procedure. Phasing and imputation using the 1000 Genomes Phase III reference panel were conducted with SHAPEIT (v2 (r904)) and IMPUTE2 (v2.3.2), respectively –30.p < 0.05. The Hardy-Weinberg equilibrium of the genotypes in the keratoconus and control groups was tested by the chi-squared test according to a p-value = 0.05. Power was calculated in G*Power software (v3.1) [We assessed the differences in allele frequencies between the control and keratoconus groups by using a two-tailed Fisher’s exact test in Prism 7  according to a threshold of e (v3.1) .2 for effect size pooling, which is the preferred method for a small number of unequally sized studies [I2 measure [We included studies conducted in European-Ancestry cohorts of keratoconus patients , 11, 14. studies . Heterog measure .The datasets used during the current study and ABI trace files are available from the corresponding author on reasonable request.n = 130) and familial keratoconus (n = 20). The main control group counted 205 unaffected control subjects from the European part of Russia . The mean age of the subjects in the main control group was greater than that in the keratoconus group to minimize the number of participants with undiagnosed keratoconus.We included 150 unrelated patients with sporadic n = 10 and famDue to the lack of genome projects including large cohorts of people from Russia to strengthen the results, we included an additional control group of 474 participants genotyped using an SNP array . This coAll 150 patients with keratoconus and 205 participants from the main control group were genotyped for three SNPs: rs1536482, rs2721051, and rs1324183. Data on the occurrence of each genotype in the keratoconus and control groups are presented in Table info metric, which estimates the accuracy of imputation, showed a high confidence score for rs1324183. Therefore, we used the imputed rs1324183 genotypes for the additional control group in the analysis. Genotyping data are presented in Table p-value < 0.05 for Fisher’s exact test). Therefore, we merged the two control groups to increase the analysis power.Two candidate SNPs, rs1536482 and rs2721051, were directly genotyped from the SNP array data for the additional control group. The IMPUTE2 p-value < 0.05) between the keratoconus and main control groups for all three SNPs: rs1536482 near the COL5A1 gene, rs2721051 near the FOXO1 gene, and the rs1324183 minor allele near the MPDZ gene . For rs1324183, the I2 value for heterogeneity was moderate – 55%.To compare the results for our cohort with those from other reported studies on keratoconus-associated loci, we carried out a meta-analysis of rs1536482, rs2721051, and rs1324183 using the genotyping data from our keratoconus group and combined control group. The results of this analysis are provided in forest plots in Fig. COL5A1 promoter  was sequenced in a subgroup of keratoconus patients with at least one rs1536482 minor allele (n = 94). We detected three SNVs with European population frequencies ≤1% according to the ALFA project: rs1043208782, rs569248712, and rs548525119  in the control groups, which may be a specific feature of the Russian population. This finding needs further investigation in larger samples.COL5A1, which encodes the collagen type V alpha 1 chain. Collagen V type is a regulatory and structural component of collagen fibrils. In the cornea, it assembles with collagen type I into heterotypic collagen fibrils. Mutations in the COL5A1 gene are the most common cause of Ehlers-Danlos syndrome [COL5A1 gene lead to a decrease in corneal thickness and the density of collagen fibrils, similar to the changes observed in keratoconus [COL5A1. Additionally, it was previously found that patients with keratoconus do not show enrichment in rare potentially pathogenic variants in protein-coding sequences, and only intron variants of unknown significance were found [COL5A1 promoter. We sequenced the COL5A1 promoter in a subgroup of keratoconus patients who had at least one rs1536482 minor allele. We identified three rare SNVs. Among these SNVs, rs548525119 did not cosegregate in relatives. The involvement of rs1043208782 and rs569248712 in keratoconus pathogenesis needs further investigation. These SNVs were present in a small proportion of keratoconus patients (two out of 94). Therefore, we conclude that rare SNVs in the COL5A1 promoter do not play a major role in keratoconus susceptibility associated with rs1536482. This is consistent with our results showing that the presence of the rs1536482 minor allele is not associated with the COL5A1 gene expression level [SNPs rs1536482, rs2721051, and rs1324183 are located in the intergenic space, so their direct association with nearby genes is uncertain. Nevertheless, we present some data on the possible role of COL5A1 in the pathogenesis of keratoconus. The nearest gene to rs1536482 with a known function is syndrome . In patiatoconus , 39. There found , 41. TheThe study is limited by its relatively modest sample size. However, the sample size examined in our study was comparable to those in other candidate gene studies of keratoconus , 11.COL5A1 gene, rs2721051 near the FOXO1 gene, and rs1324183 near the MPDZ gene were significantly associated with keratoconus in a Russian cohort. We found two rare variants in the COL5A1 promoter, one of which, rs569248712, cosegregated with the phenotype in the analyzed family.We found that rs1536482 near the


This systematic review determined the reported treatment strategies, their individual success rates, and other outcome parameters in the management of critical-sized bone defects in fracture-related infection (FRI) patients between 1990 and 2018.A systematic literature search on treatment and outcome of critical-sized bone defects in FRI was performed. Treatment strategies identified were, autologous cancellous grafts, autologous cancellous grafts combined with local antibiotics, the induced membrane technique, vascularized grafts, Ilizarov bone transport, and bone transport combined with local antibiotics. Outcomes were bone healing and infection eradication after primary surgical protocol and recurrence of FRI and amputations at the end of study period.Fifty studies were included, describing 1530 patients, the tibia was affected in 82%. Mean age was 40 years (range 6–80), with predominantly male subjects (79%). Mean duration of infection was 17 months (range 1–624) and mean follow-up 51 months (range 6–126). After initial protocolized treatment, FRI was cured in 83% (95% CI 79–87) of all cases, increasing to 94% (95% CI 92–96) at the end of each individual study. Recurrence of infection was seen in 8% (95% CI 6–11) and amputation in 3% (95% CI 2–3). Final outcomes overlapped across treatment strategies.Results should be interpreted with caution due to the retrospective and observational design of most studies, the lack of clear classification systems, incomplete data reports, potential underreporting of adverse outcomes, and heterogeneity in patient series. A consensus on classification, treatment protocols, and outcome is needed to improve reliability of future studies. Segmental bone loss after trauma remains a challenging problem for orthopedic trauma surgeons. When a bone defect exists combined with fracture-related infection (FRI), the chances of successful bone consolidation and clearance of infection are reduced.A ‘critical-sized’ defect is a bone defect which is not expected to heal in the absence of a secondary  intervention. There is no agreed definition of what constitutes a critical defect in humans. Court-Brown defined it  as a defOver the past decades, critical-sized bone defects in FRI have been treated using different protocols. Techniques used  all have different indications and success rates, resulting in a wide range of clinical outcomes. The aim of this systematic review and pooled analysis was to evaluate identified treatment strategies, their individual success rates and other outcome parameters regarding critical-sized bone defects used between 1990 and 2018.A literature search was completed with the help of a biomedical information specialist on June 25, 2018, using Medline, Embase, Web of Science, Cochrane, and Google Scholar. The search strings are provided in Appendix General information of all studies .Data from surgical protocols .Clinical outcomes .Data from each included study were extracted by two authors independently (HB an LWV). Disagreements were discussed until agreement was reached. Data were collected in three areas.http://www.medcalc.org; 2017) was used for pooling binominal data. Heterogeneity was quantified using the Cochran’s Q test and I2 statistic. When I2 was < 40%, a fixed effects model was used, and a random effects model, when I2 was ≥ 40%. Pooled estimates are reported with their 95% confidence intervals (CI). Publication bias was assessed from funnel plots for each clinical outcome and per treatment type separately. The majority of studies only provided a mean but not the standard deviation. Thus, a full meta-analysis for continuous data was not feasible. Continuous data were pooled by calculating the weighted mean using Microsoft Excel. Sample size of the individual studies was used as weighting factor. The pooled mean is reported with the range.A quality assessment of all included studies was done according to the revised and validated versions of MINORS . ResultsAfter selection, 43 studies –50 were Nine series (18%) described treatment of bone defects by cancellous grafts alone and nine series (18%) by the use of a cancellous graft in combination with local antibiotics, either beads or an antibiotic rod. The induced membrane technique was described in eight (16%) series, and treatment with a vascularized bone graft was used in four series (8%). Bone transport alone was used in 15 series (30%), and bone transport in combination with local antibiotic beads in 5 (10%).Table In 18 studies , 44, 49 The 9 studies (18%) describing treatment with a cancellous graft only included 311 (20%) patients, with a mean age of 40 years (range 16–68) and a mean bone defect of 4.6 cm (range 1–16). Four studies (44%), used a one-stage reconstruction, three studies (33%) a two-stage, and one study (11%) a three-stage. One study (11%) did not describe the number of stages.The other 9 studies (18%), which used a cancellous graft after placement of PMMA beads or a cement rod, described the treatment of 161 (11%) patients. The mean patient age was 37 years (range 18–79) and the mean bone defect length was 4.9 cm (range 1–12). The majority, 67%, were treated by a two-stage protocol, two studies (22%) used a three-stage protocol and one (11%) did not mention the number of stages. Mean time between placement and removal of the antibiotic carrier was 31 days.Eight series , 43, 48 In four (8%) studies , 41, 46,In 20 studies , 47, 50 The remaining five studies (26%) used a combination of bone transport and local antibiotics to treat 164 (11%) patients, aged 43 years (range 15–68), with a mean bone defect of 5.7 cm (range 3–14). All studies used local antibiotics in the form of beads, four studies (80%) used PMMA as a carrier in a two-stage design, and one (20%) used calcium sulphate pellets in a one-stage protocol. Mean time before removal of the PMMA beads was 42 days.After initial protocolized treatment, FRI was cured and bone defects healed in 83% (95% CI 79–87) of all cases, increasing to 94% (95% CI 92–96) with further treatment, at the end of the total study period. Recurrence of infection was seen in 8% (95% CI 6–11), and amputation in 3% (95% CI 2–3) of all cases.Figure Figure Adequate debridement of non-viable bone remains one of the cornerstones of the management of FRI , 52. ThiThis systematic review revealed that the current most popular techniques resulted in comparable rates of healing and recurrence of infection. It is however unlikely that one reconstruction technique will be able to address all types of bone defects in FRI patients. Kadhim et al.  reviewedDue to several factors, it was not possible to draw firm conclusions from the data in this regard. Firstly, the heterogeneity of the patient populations made comparison of outcomes difficult. There was, for example, a difference in the mean size of the defects among the different treatment groups. Series describing the use of vascularized grafts or bone transport techniques treated larger bone defects than series using cancellous grafts. Also, in studies describing a two-stage reconstruction protocol, often more surgical revisions were performed than initially expected.The choice of reconstruction technique depends on many factors. These include the host’s physiological status and ability to participate in the rehabilitation program, the shape and location of the defect, duration of the defect , quality of the surrounding soft tissue, bone quality, the presence of deformity, adjacent joint contracture/instability or limb length discrepancy, as well as the experience of the surgeon . UnfortuMost studies provide a cohort of cases treated with a single technique, rather than a consecutive series of patients, selected because of the characteristics of the disease . This maSecondly, of all included series, 12 (24%) , 46, 47 The current lack of a universally accepted classification system for both FRI and post-infective bone defects is evident in this systematic review. The need remains for the development of a pragmatic classification system that can be used, not only to guide treatment, but also to adequately characterize the patient cohort for research purposes –62. SimiIn addition to the lack of an applicable classification of post-infective bone defects, a working definition of defects is needed which cannot be expected to heal without intervention. While the difference between a stable cavitory (contained) defect and an unstable defect may be apparent, there may be a need to further define critical unstable defects to investigate the outcomes of treatment modalities in different types of critical bone defects. This definition, together with a pragmatic classification would allow the development of a practical algorithm for management of these infected defects. It may be foreseen that a 4 cm conical  humeral defect with 20% cortical bone contact may be treated differently than a 4 cm segmental (complete) tibial defect, for example .In addition to the lack of uniformity in terms of patient population, numerous other factors prohibit direct comparison of results from the studies included in this systematic review. Due to the retrospective and observational nature of most studies, treatment strategy selection was not always clearly reported and may not have been consistent. Furthermore, data were not always sufficiently complete to allow comparison of outcomes. For continuous outcomes, most studies failed to report the standard deviation along with the mean value. Underreporting of adverse effects is of particular concern, especially if risks associated with a specific treatment option in a specific clinical setting should be taken into account when choosing a reconstructive option. When looking at the asymmetrical form of the funnel plots created for all clinical outcomes, this could be the case, as is discussed above.Mauffrey and Hak  have recIt is unlikely that one single, high volume center is able to adequately study the problem of FRI. Even in such referral centers the number of patients treated in a reasonable time span is not sufficient to achieve enough statistical power to draw firm conclusions on current treatment standards and new therapeutic options. This implies that  collaboration is essential to be able to pool treatment results from individual hospitals into (prospective) clinical studies and subsequently into meaningful meta-analyses. Only then, can adequate progress in the treatment of FRI be made. To facilitate such a collaboration, uniform definitions for classification, diagnostic and treatment protocols and follow-up are required. The current meta-analysis clearly shows that the way in which data have been collected and reported in the past cannot inform best practice in the future.This is the first extensive review of bone defect treatment protocols for FRI. Six individual treatment protocols for FRI treatment were identified, i.e., cancellous grafts with or without the use of local antibiotics, the induced membrane technique, the use of vascularized grafts, or the Ilizarov bone transport technique with or without the use of local antibiotics. All show comparable outcome. Overall published work showed a high success rate of 94%, a recurrence rate of 8%, and low amputation rate of only 3%. However, data did not allow a reliable comparison across treatments, or a recommendation on which treatment strategy is appropriate for any particular clinical scenario. The results should thus be interpreted with caution due to the retrospective design of most studies, the lack of clear classification systems, incomplete data reports, underreporting of adverse outcomes, and heterogeneity in patient series. Secondly, this review reveals the true scientific and clinical needs: uniform definitions on terms used, a consensus on classification, structured treatment protocols, and clear outcome parameters are needed to improve reliability of future multicenter studies.


Phaseolus vulgaris L.) lines to acid soils and high temperature conditions in the Amazon region of Colombia is limited. In this study, we evaluated the differences among 41 common bean lines in energy use, leaf cooling, photosynthate partitioning to pod formation and grain filling, and grain yield over two seasons under acid soil and high temperature stress in the Amazon region of Colombia. Common bean lines evaluated included medium and large seeded interspecific lines of Mesoamerican and Andean gene pools with different levels of adaptation to abiotic stress conditions and some lines are improved for iron and zinc (biofortified) concentration in seeds. We found three bean lines  that were superior in their photosynthetic response, leaf cooling, photosynthate partitioning ability to pod formation and grain filling, resulting in grain yields exceeding 1900 kg ha−1 under acid soil and high temperature stress conditions. The superior photosynthetic performance was attributed to the efficient use of absorbed energy on the electron level in thylakoids, which is mainly oriented to a higher quantum yield of PSII (ΦII), lower energy dissipation in the form of heat (ΦNPQ), high linear electron flow (LEF) and high fraction of PSI centers in open state (PSIopen). We speculate that these photosynthetic and photosynthate partitioning responses of superior bean lines are part of the genetic adaptation to acidic soils and high temperature stress conditions. Among the evaluated bean lines, three lines  combined the desirable attributes for genetic improvement of stress tolerance and biofortification. These lines can serve as parents to further improve traits (energy use efficiency and multiple stress resistance) that are important for bean production in the Amazon region.Knowledge of the physiological basis for improved genetic adaptation of common bean ( Phaseolus vulgaris L.) is one of the five cultivated species of the genus Phaseolus, and it is ranked as one of the main grain legumes after soybean and peanut [The common bean  [Beans have been classified into two major gene pools based on seed size differences, with large-seeded beans being Andean (South America) and small-seeded beans being Mesoamerican  in their origin ,22,23, gneus L.) ,26. Thesneus L.) . Andean neus L.) . Improveneus L.) ,28. Thesneus L.) ,29. http://www.photosynq.org (accessed on 26 October 2021)) has been used to make a large number of these non-invasive measurements in a short period of time and this tool has been widely used for phenotyping in different crops [In the Amazon region, previous research resulted in the identification of common bean genotypes that are adapted to low fertility acid soils  and highnt crops ,36,37,38The main objective of the present study was to evaluate differences among the medium and large seeded Mesoamerican and Andean common bean lines in energy use, leaf cooling, photosynthate partitioning to pod formation and grain filling, and grain yield over two seasons under acid soil and high temperature stress in the Amazon region of Colombia. We tested the hypothesis that the well-adapted common bean lines will combine greater energy use and leaf cooling with greater partitioning of photosynthates to pod formation and grain filling under acid soil and high temperature stress. −1 with a mean value of 791 ± 93 kg ha−1. Lines GGR 147, SMG 21 and SMG 12 had higher yields, exceeding 1900 kg ha−1   presented higher number of pods per area (−2.Grain yield (GY) of the 41 evaluated genotypes ranged from 157 to 2152 kg ha kg ha−1 . Howeverper area . Among tp < 0.001). For the mobilization of pod wall assimilates toward grain formation and grain filling as reflected by PHI values, lines such as SMG 29, GGR 150, SMG 27 and GGR 147 stood out with values of more than 60% (p < 0.001). For the seed number per area (SNA), the genotypes SMG 21, GGR 47 and SMG 12 stood out for producing more than 500 seeds m−2 (p < 0.001). Lines such as SMG 4, SMG 1, SMG 10 and SMG 31 presented values lower than 100 seeds m−2. The average weight of 100 seeds was between 21.3 and 33.1 g, with lines GGR 150, GGR 147, SMG 27, DAB 295 and SMG 29 showing values greater than 30 g (p < 0.001).In relation to the mobilization of photoassimilates from vegetative structures to pod formation, the PPI values ranged from 17.2 to 78.3% , with SMthan 60% . The harthan 60% , where op < 0.001, p < 0.001, Days to flowering, in the two seasons in which the adaptation of different bean lines to acid soils and high temperature stress was evaluated, ranged from 29 to 34 days after planting (DAP). Lines such as SMR 190, GGR 149 and SMG 28 were earlier to flower as a stress response . Lines sp < 0.001). In general, all lines evaluated were able to adjust leaf temperature below the ambient value. For example, lines such as SMG 27 and SMG 23 presented more negative LTD values indicating marked leaf cooling under high temperature stress. However, other lines such as GGR 148 presented less negative LTD values and their energy use efficiency related to photosynthetic processes was also low. For LEF parameter, line SMR 168 presented the highest value while SMG 10, SMG 22 and SMR 168 presented higher values for both ECSt and vH+ parameters. Line SMR 190 showed the highest value for PSIact (p < 0.001). For example, higher values of GY were related to higher values of PSII quantum yield (ΦII), a situation presented with two lines, SMG 12 and SMG 21, and these lines also exhibited lower value of ΦNPQ  showed a higher electron transport rate combined with a higher proportion of open PSI centers (LEF = 226.2)  and higher ΦNO values  b. The opO values . In addiO values b. In terms of energy use, the bean lines, on average, used 48.1% and 30.7% for photosynthetic processes (ΦII) and dissipation of energy in the form of heat (ΦNPQ) and the remaining in unregulated processes (21.2% ΦNO) . When wep < 0.05), PPI , PHI , PNA , and SNA  (p < 0.05), HI , PNA  and SNA   was negatively related to different biomass partitioning indices  and to GY, as well as LTD . For exa < 0.05) . Similar < 0.05) . In gene < 0.05)  while ET < 0.05) .When analyzing the relationship between the partitioning indices and the functioning of the photosynthetic apparatus, we found very important results in relation to the adaptability of the different bean lines under conditions of acid soils and high temperatures. For example, leaf cooling (LTD) influenced the number of grains  as well as phenological adjustment  and pollen viability  . At the Phaseolus for improving stress tolerance and seed size and marketable color. Previous research by Suárez et al. [In crops such as the common bean, where grain is the product of interest, the main selection criteria are focused on agronomic performance, which is reflected through genetic improvement in grain yield . This stz et al. ,11 indic2 fixation capacity, nutrient assimilation, and water use under heat stress conditions [−1) but their grain yield values were not that high which is likely due to increased sensitivity to high temperatures that impacted pod formation and grain development. Only two lines, SMG 4 and SMG 28 showed higher values of HI (>0.5) and among these two lines, the latter was early maturing with GY value higher (813 kg ha−1) than the average (791 kg ha−1).High-yielding lines such as SMG 12 and SMG 21 showed higher values of CB compared to other lines. This may be due to their greater ability in net COnditions . Other lAn important trait for improving the adaptation of common bean lines under stress conditions is the ability to redistribute stored assimilates to grain ,40. SeveFour lines  stood out for maintaining PHI values greater than 60%, this being one of the main physiological traits of importance on photoassimilate remobilization toward better grain filling ,14,15,44Grain yield values contributed to identify six significantly different genotypes  in terms of sensitivity to stress. This means that in this study, the negative effect of environment was big and/or the selected lines are generally not well adapted to Amazon conditions. Higher correlation of GY to pollen viability and canopy biomass also support the idea of selection in a harsh-environment. Usually, PV and CB values have either a low or not significant effect on grain yield . HoweverWe found that some genotypes were smaller earlier  with shorter time taken to reach flowering . From these lines, only SMG 26, 28, 30, and SMR 190 also had a shorter (not significantly) number of days to physiological maturity (DMP ≤ 74). From the latter group, with four lines, we observed that this earliness negatively impacted the values of PNA and SNA  suggesting that these lines have less time for photosynthate remobilization from vegetative parts to the pod wall (PPI) and to the seed  ,46. Line−1, which was significantly lower than the average (791 kg ha−1). It has been observed that high temperature stress during pre-flowering and flowering stages causes a malformation of pollen, which decreases its viability and generates flower abortion that later affects the ability to produce pods and grains ,51,52,53On the other hand, lines such as GGR 146 and 147, and SMG 12, 14, and 21 showed PV values that were significantly lower than the average but their GY values were higher. Interestingly, both GGRs were earlier to flower; therefore, the reduction in PV values in these lines might have resulted from the earlier effects of high temperature on anther indehiscence , degenerWe previously described how high temperatures affect phenological development as well as grain yield in bean lines and the different mechanisms that contribute to cope with high temperature stress in Amazon ,11,31. ISimilarly, two lines (SMG 12 and SMG 21) showed adaptive physiological mechanisms to the heat stress condition. Based on the data obtained from the correlation of the different physiological and environmental variables, we found that the decrease in LEF and ECSt (ATP synthase capacity), influenced LTD value and this could be attributed to a slight increase in abscisic acid (ABA) level leading to a higher stomatal closure . These tThe less negative LTD (closer to zero) in some genotypes is likely connected to lower transpiration rate . Since tWe hypothesize that under combined acid soil and high temperature stress, bean lines activated different mechanisms to dissipate heat and keep photosystems unaffected or efficiently acclimatized by increasing transpirational flow ,32,36. FBased on different mechanisms observed in this study, we highlight the adaptive responses of lines SMG 12, SMG 14, SMG 21, SMG 27, and GGR 147 to the combined stress of acid soil and high temperature. These lines may be well adapted to higher temperature environments and may also be considered as progenitors in breeding programs focused especially on the selection of materials that combine a better physiological response in relation to carbon fixation and with a greater capacity for dry matter partitioning towards pod development and grain filling under heat stress conditions. The adaptation of individual genotype depends on its responses and degree of susceptibility to a stress condition. In this study, we observed that some lines have the characteristic of producing abundant biomass but with low grain yield ,62. Corr2 for grain formation and filling. This behavior has been previously observed by Polania et al. [In this study, we found that genotypes that tend to form larger seeds show greater sensitivity to stress, mainly in their capacity to remobilize assimilated COa et al.  in genotWe highlight the high efficiency for grain formation in lines such as SMG 12 and SMG 21 that tend to be superior to lines such as SMR 180 and BFS 81 . Thus, the variables of GY, PHI and PPI were potential contributors to phenotypic differentiation at the genotype level in the common bean accessions evaluated in this study. The main characteristic evaluated in this study is related to the medium and large grain size of the Mesoamerican gene pool. In addition to the above, there are other characteristics that these lines have which are related to high mineral content as well as tolerance to drought and heat. For example, the GGR lines were selected based on 100-seed weight that exceeds 36 g and the GGR 146 (GGB 13 × SMR 191) line had within its progeny materials of large grains (GGB) with resistance to drought with high mineral content (SMR) while the GGR 147 (SMC 214 × (GGR 1 × SMC 232)F1) had in its cross a 75% contribution in drought resistance and mineral traits and the remaining in the size of the grain. The SMR (SMR 189 × (SMC 199 × BFS 81)) line is from gamete selection with resistance to angular spot, and tolerant to soils with low fertility and drought conditions. It was derived from a triple cross where SMR 189 provides high mineral content and adaptation to drought and good plant growth, while SMC 199 is a line that provides resistance to drought and high mineral content and good plant growth; finally, BFS 81 with tolerance to low fertility and drought. The BFS 81 line contributes 25% of the cross in terms of tolerance to acid soils and low fertility in the Amazon, a condition that has been previously reported by Suárez et al. . FinallyBased on the results from this study, three lines  were identified and these three lines combined the desirable attributes for genetic improvement of stress tolerance and biofortification in common bean. These three lines can serve as parents to further improve traits (energy use efficiency and multiple stress resistance) that are important for bean production in the Amazon region.−1. The average temperature is 25.5 °C and relative humidity is 84%. During the growing season the maximum and minimum air temperatures were 36.8 °C and 17.4 °C (season 1), 36.6 °C and 18.4 °C (season 2), respectively  of 2.58 mg kg−1, total base saturation of 7.1% (cmol(-) kg−1 : Ca, 0.38; Mg, 0.1; K, 0.14; Na, 0.1; total bases: 0.8), a cation exchange capacity of 11.3 cmol(-) kg−1, and an exchangeable Al content of 6.3 cmol(-) kg−1 with 73.4% Al saturation.Two field trials were conducted at the Macagual Research Center of the University of Amazonia in Florencia, Colombia, located at 1°37′ N latitude and 75°36′ W longitude and an altitude of 250 meters above sea level. The first trial was conducted during December 2019 to February 2020, and the second trial was conducted during June to August 2020. The site has a tropical rainforest climate, with an annual rainfall of 3800 mm and 1700 h of sunshine per yearectively . The totectively . Soil sa−2).For the study, 41 bean genotypes belonging to the Mesoamerican and Andean gene pool were selected: 27 advanced common bean lines interspecific mesoamerican common bean lines ; four advanced biofortified lines ; three advanced common bean lines from the Andean gene pool ; seven large seeded lines . DAB 295 was used as a control genotype. All lines were provided by CIAT and the main characteristics of the lines are shown in 2) per plot. A total of four plants were sampled to determine canopy biomass (CB). Similarly, at the time of harvest, another destructive sampling was performed , and the dry weight of leaves, stems, pods and seeds was recorded. The dry weights of the samples from two samplings, the number of seeds per area (SNA), and the number of pods per area (PNA) were determined. The following dry matter partitioning indices were determined according to Beebe et al. (2013). Pod partitioning index (PPI): dry weight of pod biomass at harvest/total canopy biomass (CB) dry weight at mid-filling × 100; pod harvest index (PHI): seed biomass dry weight at harvest/pod biomass dry weight at harvest × 100; and harvest index (HI): seed biomass dry weight at harvest/total canopy biomass dry weight at mid-pod filling × 100. We estimated HI and PPI using the CB value at mid-pod filling growth stage assuming that this is the time that reflects the maximum shoot vigor of the genotype [−1). The weight (g) of 100 seeds was quantified with a homogeneous random sample. During the mid-pod filling growth stage , a destructive sample was collected from a 0.5 m row segment  was recorded for each genotype, this being the number of days after planting in which 50% of the plants have developed at least one flower. Likewise, the number of days to physiological maturity (DPM) was determined for each genotype, being the number of days after planting in which 50% of the plants had at least one pod that lost its green pigmentation. To determine the influence of high temperature on the different bean lines, pollen viability (PV) was evaluated following the methodology of Porch and Jahn . Flower Data related to environmental variables and photosynthetic traits based on chlorophyll fluorescence were collected using the default protocol Photosynthesis RIDES in which the measurement starts automatically once the clamp is open and closed. This protocol was developed by PhotosynQ for MultispeQ hand-held device . We usedAmong the environmental variables, humidity and ambient temperature were measured, and the difference between the latter and the leaf temperature was used to calculate the leaf temperature differential (LTD). The linear electron transport (LEF) was estimated using the equation LEF = Φ(PAR)-YϕII, where f is the fraction (f = 0.45) of absorbed radiation which is transferred to the PSII centers and ϕII corresponds to the effective quantum yield . LEF is The proton conductivity of the thylakoid membrane (gH+) and the amplitude of electrochromic bandshift signal (ESCt) were determined by the dark-interval relaxation kinetic of electrochromic shift (DIRK method ). The meWe measured relative chlorophyll content (SPAD), the quantum efficiency for photosystem II photochemistry (ΦII), photoprotective non-photochemical quenching (ΦNPQ), and the basal dissipation of light energy (ΦNO) using the MultispeQ . ΦII indThe quenching due to non-photochemical dissipation of absorbed light energy (NPQt) was calculated according to Tietz et al. . LikewisAccording to the information reported in the PhotosynQ platform, the total number of data obtained by the MultispeQ device during sampling was 878, in which there were no errors. Fluorescence outliers were identified and excluded from the analysis using Pearson’s standardized residuals. This type of residual provides a measure of how well the observation is predicted by the model, by recording those data that do not present an adequate fit .To determine the differences between bean lines in relation to the different variables of agronomic performance and chlorophyll fluorescence, a Generalized Linear Model (GLM) was fitted. The bean line was selected as a fixed factor and the blocks nested within the growth stage of evaluation in each season and the plots associated with the bean lines within the blocks were included as random effects. The assumptions of normality and homogeneity of variance were evaluated using an exploratory analysis of residuals. To determine the differences between evaluated bean lines, Fisher’s post hoc LSD test was used with a significance of α = 0.05. The association between agronomic, phenological, environmental and chlorophyll fluorescence-related variables was analyzed by calculating Pearson’s correlation coefficients. Different scatter plots were made to determine the lines with the best adaptation to acid soils and high temperature stress. On the Y axis of the scatter plots were placed variables of agronomic performance such as grain yield as well as variables related to energy use such as ΦII, ΦNPQ, and ΦNO and those affecting these variables were plotted on the X axis which corresponded to CB, PPI, PHI, ΦII, LEF and LTD. The variables located in the scatter plots were associated with the effect of the gradient of different variables. Their intensity is identified with the change in the color  and as well as the magnitude that related to circle size. The graphs were visualized in four quadrants by plotting the corresponding means on each axis. For the Generalized Linear Model (GLM) analysis, we used the lme function of the nlme package , both GL−1 under acid soil stress and high temperature conditions. The heat stress tolerance of the different lines evaluated was attributed to the efficient use of the energy absorbed at the electron level in the thylakoids, which was mainly oriented to a higher quantum yield of PSII (ΦII), coupled with a lower dissipation of energy as heat (ΦNPQ), as well as a high linear electron flux (LEF) and a high fraction of PSI centers in the open state (PSIopen). These physiological changes translated into the ability to produce greater canopy biomass, phenological adjustments in relation to days to flowering and maturity as well as the efficiency in mobilizing photoassimilates to produce more pods and grains. These three  physiologically efficient lines combined desirable attributes for genetic improvement of stress tolerance and biofortification and, therefore, can serve as parents to further improve the traits (energy use efficiency and resistance to multiple stresses) that are important for bean production in the Amazon region.Based on physiological evaluation of the energy use, leaf cooling, photosynthate partitioning for pod formation, filling and grain yield of the different bean lines, we found that three bean lines  were superior in their photosynthetic response resulting in grain yields that were above 1900 kg ha


Copy-number variants (CNVs) are an important cause of human neurodevelopmental disorders. We present the first case of a 424 kb de novo 20q13.11q13.12 microdeletion in a patient with attention deficit disorder, tics and autistic behaviors, such as emotional and behavioral problems, and movement stereotypes. This region includes three genes expressed in the brain: SFRS6, PTPRT and L3MBTL. Our results suggest that loss of the chromosomal region 20q13.11q13.12 is causative for the clinical findings observed in the patient. Neurodevelopmental disorders are a group involving developmental impairments of the central nervous system. These include intellectual disability (ID), autism spectrum disorders (ASDs), attention deficit hyperactivity disorder (ADHD), impaired motor function, learning, language or non-verbal communication and tics . EpidemiThe patient was born to non-consanguineous parents at 40 Hbd weeks of gestation by caesarean section. Body parameters were within the normal range with birth weight of 3250 g, body length 58 cm and head circumference 34 cm. Early developmental milestones were normally attained. At school age (from age 6) he was noted to have stereotyped movements, nervous tics, social and emotional disturbances, and attention deficit disorder. Currently – at age 13 − he still manifests nervous tics in stressful situations. He has been diagnosed with dyslexia, dysgraphia, attention deficit disorder and continues to have learning difficulties. His intelligence is assessed at average level. No significant dysmorphic characteristics have been observed aside from a slightly asymmetric and narrow chest, small ears, and synophrys. EEG analysis showed discrete changes on the right temporal area. The family history is positive for Asperger syndrome in his father’s brother, depression in his mother and schizophrenia in his paternal grandmother.Oligonucleotide array comparative genomic hybridization (aCGH) was performed using 8x60K from Oxford Gene Technology (CytoSure ISCA v2). Array CGH has genome-wide coverage with an average resolution of 120 kb. DNA digestion, labeling, and hybridization were performed following the manufacturer’s instructions.SFRS6 , L3MBTL  and the first exon of the PTPRT gene . Both parents were tested by aCGH and were negative for the aberration, indicating that the 20q13.11q13.12 microdeletion occurred de novo in the patient . The deleted region contained three genes:  patient .PTRP gene in a patient with neurodevelopmental disorder.We performed a literature review and database search for individuals carrying a microdeletion involving the long arm of chromosome 20. To date, there are very few medical reports of 20q13 deletion, most of which involve regions more distal to our case , 5. We pPTPRT, SFRS6 and L3MBTL expressed in many tissues, mostly in the brain. Two of these genes (PTPRT and L3MBTL) are haploinsuffcienct (sensitive to haploinsuffciency). The PTPRT gene is the candidate gene in the 20q13.11q13.12 region most likely associated with the pathology identified in our patient. Receptor protein tyrosine phosphatase (PTPRT) is a transmembrane protein that interacts with other cell adhesion molecules and regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. The PTPRT protein is a regulator of synaptic formation and neuronal development [PTPRT in hippocampal neurons correlates with synaptic formation and dendritic arborization. On the other hand, knockdown of PTPRT decreased neuronal transmissions and attenuated neuronal development [PTPRT was identified as a potential candidate gene for autism spectrum disorders (ASDs). A genome-wide association study found suggestive linkage results between the chromosome region 20q11.21 and ASDs [PTPRT. Further, Christian et al. identified an inherited duplication including this gene in a patient with ASDs [The deleted region observed in our patient includes three genes: and ASDs . One of ith ASDs .PTPRT gene. They observed only a slight change in locomotor activities and anxiety-related behaviors in the homozygous knocking mice with PTPRT mutation, and higher social approach scores in male homozygous mice. It seems that PTPRT phosphatase function affects mouse social behaviors which are similar to ASDs patients’ symptoms [PTPRT, in siblings with intellectual disability [et al. found 7 contiguous single nucleotide polymorphisms at chromosome 20q12q13.12 in all the affected individuals [PTPRT is a candidate gene for neurodevelopmental disorders, such as ADHD and ASDs.A large group of the genes identified as risk factors for ASD plays an important role in synaptic homeostasis . This insymptoms . Moreovesability . Furtherividuals  from a sSFRS6. The protein encoded by this gene belongs to the splicing factor SR family and has been shown to bind with and modulate another member of the family − SFRS12. SFRS6 has been found mainly in the brain. There are only several publications about the SRSF6 gene – it is proposed as a candidate gene for colorectal tumors [The second gene deleted in our patient is l tumors  and is al tumors .L3MBTL gene is also mainly expressed in the brain. This gene encodes a member of the polycomb proteins’ family which causes chromatin condensation and repression of transcription [The cription . This prcription .PTPRT gene suggests that haploinsufficiency of this gene may result in the clinical features observed in our patient: stereotype movements, nervous tics, social and emotional disturbances and attention-span deficits. Little is known about the SFRS 12 and L3MBTL genes, but in view of the fact that these genes are expressed mainly in the brain, we suspect that their deletion may also contribute to the pathogenesis of the developmental disturbances in our patient.In summary, the established function of the


Introduction: Various types of arrhythmia have been reported during cesarean section under spinal anesthesia. But the possible causative factors and the effects of arrhythmia on immediate post-delivery neonatal outcome are not well established.Methods: This prospective observational study was conducted over a period of one year in a tertiary care hospital on women undergoing cesarean section under spinal anesthesia. The objectives of the study were to determine the incidence of arrhythmia, its types, the possible factors influencing arrhythmia, and the immediate post-delivery neonatal outcome. Data collected were analyzed using Statistical Package for the Social Sciences (SPSS) software version 21 .4 level and dose of intrathecal hyperbaric bupivacaine was more than 2.2 mL . Also, uterine manipulation led to sudden bradycardia and transient cardiac asystole in two patients which was preceded by subjective symptoms of pain and discomfort. None of the neonates required cardiopulmonary resuscitation or neonatal intensive care unit admission within an hour of birth. APGAR (Appearance (skin color), Pulse (heart rate), Grimace (reflex irritability), Activity (muscle tone), and Respiration) scores at 1 and 5 minutes were similar in all the newborns born to mothers with or without arrhythmia.Results: In our study, the incidence of arrhythmia was 31.9% during cesarean section under spinal anesthesia; and sinus bradycardia was the most common type. Arrhythmia occurred more in women with hypotension, when maximum block height was above T4 and T6, using lower possible drug dose to provide adequate level of sensory block, prompt management of hypotension, and strict monitoring during uterine manipulation may reduce the overall incidence of arrhythmia. Intraoperative arrhythmia, however, does not adversely affect the immediate post-delivery neonatal outcome.Conclusion: The occurrence of arrhythmia during cesarean section under spinal anesthesia, though very common, is rarely life-threatening. Keeping maximum level of block height between T Obstetric anesthesia is one of the areas of anesthetic practice where an anesthesiologist faces many challenges owing to the care of both the mother and fetus simultaneously.6. Block height may be the single most important variable contributing to the occurrence of new conduction delays in otherwise healthy women [Cesarean section can be performed under general anesthesia, epidural anesthesia, or spinal anesthesia . Howeverhy women .The etiology of arrhythmia during cesarean section under spinal anesthesia is multifactorial. The effect of arrhythmia and hemodynamic alterations on maternal and immediate neonatal outcome is not yet clear.This study was thus conducted to evaluate the incidence of different types of arrhythmia during cesarean section under spinal anesthesia and to identify the associated risk factors and its effects on immediate post-delivery neonatal outcomes. The knowledge acquired would help us modify the potential predisposing factors.This prospective observational study was conducted over a period of one year from October 2019 to September 2020 in a tertiary care hospital of north-eastern India. Institute ethics committee approval was taken before commencing the study. All pregnant women of the American Society of Anesthesiologists (ASA) physical status I and II, irrespective of age, weight, and height planned for elective or emergency cesarean section under spinal anesthesia were included in the study. Any women with preoperative electrocardiographic (ECG) abnormality, electrolyte disturbance, cardiorespiratory, renal, hepatic, or thyroid disease, pregnancy-induced hypertension, or any other condition or medications that may influence the occurrence of arrhythmia were excluded from the study. Patients who required conversion to general anesthesia were withdrawn from the study. Exclusion criteria also included cases with any signs of fetal distress and fetus with congenital anomaly.3-L4 interspinal space using a 25 gauge spinal needle. The patients were then positioned supine with left uterine displacement till the appropriate block height was achieved following which the surgeons were notified to begin the surgery. Oxygen at the rate of two liters per minute via nasal prongs was administered until delivery was accomplished. Ringer’s lactate was administered to all the mothers intraoperatively.Informed written consent was obtained from parturients who were recruited in the study. All study participants were preloaded with 500 milliliters of Ringer’s lactate through an 18 gauge intravenous cannula. ECG, non-invasive blood pressure (NIBP), and oxygen saturation (SpO2) were monitored intraoperatively and continued for an hour postoperatively. All women were given spinal anesthesia in the left lateral position following the aseptic technique. Intrathecally, 2 to 2.6 mL of 0.5% hyperbaric bupivacaine was administered at the LLevel of block height was assessed after 5, 10, 15, and 20 minutes of spinal anesthesia (by pin-prick sensation). Blood pressure was recorded at 5 minutes interval. Hypotension, a decrease in mean arterial pressure (MAP) by more than 30% was noted. Also, treatment with intravenous mephentermine or ephedrine, if required, was noted along with the dosage. Uterine manipulation  was also noted.All episodes of arrhythmia were recorded and correlated with the maximum level of block height, dose of intrathecal bupivacaine used, uterine manipulation, and hypotension. Block height at the time of arrhythmia and any intervention required to terminate the event were also recorded.After delivery, neonatal parameters were recorded in terms of APGAR score at 1 and 5 minutes, need for cardiopulmonary resuscitation (CPR), and neonatal intensive care unit (NICU) admission in all the mothers with or without arrhythmia and correlated.All the data collected were entered in Microsoft Excel  and analyzed using Statistical Package for the Social Sciences (SPSS) software version 21 . Continuous variables were analyzed with independent Student’s t-test and the categorical variables compared using Chi-Square test. A P value less than 0.05 was considered statistically significant.The results of the study are tabulated in Tables  A total of 567 cesarean sections were performed during the study period, of which 216 pregnant women fulfilling the inclusion criteria were included in our study. Out of these 216 women, 69 women (31.9 %) had occurrence of different types of arrhythmia during cesarean section under spinal anesthesia.Women in both the arrhythmia and non-arrhythmia group were similar with respect to their age and body mass index (BMI) Table .Among the different types of arrhythmia, the most common was sinus bradycardia n=42; 60.9%) followed by sinus arrhythmia , premature atrial contractions , premature ventricular contractions , supraventricular tachycardia , and Mobitz type I   (Table 4 (Table Women with maximum block height above T1) Table . Also, s1) Table . More th 4 Table .Hyperbaric bupivacaine (0.5%) was used in all cases though the doses varied ranging from 2 mL to 2.6 mL. 25.5% of patients who received doses up to 2.2 mL had arrhythmia; however, 43.03% of patients who received dose more than 2.2 mL had some type of arrhythmia and the difference was statistically significant     in seven cases. Block height attained were T1) Table .Hypotension was recorded in 79.6% of cases (172 out of 216), lasting for 168 seconds to 12 minutes in total duration. In 43.47% (30 out of 69) cases, arrhythmia was preceded by hypotensive episodes, of which 18 were sinus bradycardia, 10 were sinus arrhythmia, and two were premature atrial contractions. A statistically significant association was found between hypotension and arrhythmia   Table . None of4 (Table 4 may inhibit cardio-accelerator fibers (T1-T4) resulting in unopposed parasympathetic effect on sinoatrial (SA) and AV node with consequent bradycardia. In our study, 18 cases of bradycardia were preceded by hypotension which can be explained by the Bezold-Jarisch reflex where reduced venous return results in reflex bradycardia. Nishikawa et. al. also reported a case where changing position from Trendelenburg to supine resulted in bradycardia and asystole secondary to reduced venous return [5 and above as an important determinant for the development of bradycardia under spinal anesthesia and in most of the cases they were benign [Spinal anesthesia is the most common type of anesthesia administered during cesarean section, although there are reports of various types of arrhythmia intraoperatively such as bradycardia, ventricular tachycardia, atrioventricular (AV) conduction block, premature contractions, etc. An incidence of arrhythmia of 31.9% was found in our study which was mostly uneventful and benign but a couple of them progressed to asystole or cardiac arrests. Sinus bradycardia was the most common type of arrhythmia (19.4%) in our study. Shen et al. reported an incidence of arrhythmia of 79.52% in patients undergoing cesarean section under spinal anesthesia which was much higher compared to our findings. However, in their study, bradycardia was present in only 6.7% of cases and one of them had loss of consciousness [8 and complained of anxiety and discomfort during manipulation. Pain, anxiety, or emotional stress may result in such episodes of vasovagal syncope. Many cases have been reported where hemodynamic instability followed placental expulsion, traction, or inversion of the uterus. Vasovagal reflex via afferent nerve stimulation due to infundibulopelvic ligament stretching may be responsible for such episodes [Uterine manipulation in the form of exteriorization is not uncommon during the cesarean section for uterine repair or inspection of the posterior wall. Two cases in our study had sudden bradycardia during uterine manipulation which progressed to asystole, however, the episodes were reversed on stopping the uterine manipulation and did not last for more than 2 to 3 seconds. Both the women had block height of Tepisodes . Proper 5 and he cited parasympathetic overactivity secondary to cardiac sympathetectomy and reduced venous return as the cause [8 and there was no uterine maneuver or complaint of pain or discomfort prior to the arrhythmic event. Compared to Mobitz type II which arises at infra-nodal level, type I block usually arises at AV nodal level with lesser chances of progression to complete heart block. Type I block may occur due to multiple organic causes, however, may also occur in 1-2% of the healthy population without any consequence. The underlying reason of this episode in our patient was not clear.Kalra et. al. reported an episode of benign, asymptomatic Mobitz type I block in a patient without any underlying heart disease or conduction block undergoing cesarean section under spinal anesthesia. The maximum block height was The cause . In our Drug dosage or volume may influence the incidence of arrhythmia secondary to higher level of block and also hypotension. By reducing the dose of an anesthetic drug, arrhythmia can be minimized but at the cost of supplemental analgesic requirement and risk of conversion to general anesthesia. Ginosar et. al. suggested against the use of low dose bupivacaine for spinal anesthesia in case of emergency cesarean section due to the higher risks of failure and delayed onset ,12. TherThe effect of different systemically administered drugs on the occurrences of arrhythmia under spinal anesthesia during cesarean section is difficult to evaluate because of multiple overlapping factors. Though there is a theoretical possibility of arrhythmia following oxytocin infusion, it is rarely reported . And at Blockade of cardio-accelerator fibers resulting from high level of block may cause hypotension and bradycardia at the same time making it difficult to predict if one is the cause or consequence of another in such clinical conditions. In 43.47% of the cases of arrhythmia in our study, hypotension has been found to precede arrhythmia which could be due to reflexes secondary to the reduced venous return and hypotension. A proper preloading or co-loading and frequent monitoring of blood pressure especially after commencement of spinal anesthesia or following position changes along with a prompt correction would help prevent the development of such events .Women with antenatal arrhythmia may have neonatal effects in terms of intrauterine growth retardation or placental abruption . However4 and T6, using a lower dose of bupivacaine and early detection and management of hypotension may reduce the incidence of arrhythmia. Also, pain and discomfort during uterine manipulations, especially when the block height recedes below T8 may lead to sudden bradycardia and cardiac asystole which can be prevented by paying attention to patient’s subjective symptoms and intense monitoring of pulse and ECG with prompt necessary intervention. The occurrence of intraoperative arrhythmia, however, does not adversely affect the immediate neonatal outcome.The occurrence of arrhythmia during cesarean section under spinal anesthesia is very common but may rarely be life-threatening. Keeping the block height between T


Commonly known as Batten disease, the neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of rare pediatric lysosomal storage disorders characterized by the intracellular accumulation of autofluorescent material (known as lipofuscin), progressive neurodegeneration, and neurological symptoms. In 2002, a disease-causing NCL mutation in the CLN6 gene was identified (c.214G > T) in the Costa Rican population, but the frequency of this mutation among local Batten disease patients remains incompletely characterized, as do clinical and demographic attributes for this rare patient population.To describe the main sociodemographic and clinical characteristics of patients with a clinical diagnosis for Batten Disease treated at the National Children's Hospital in Costa Rica and to characterize via molecular testing their causative mutations.DNA extracted from buccal swabs was used for CLN6 gene sequencing. Participants’ sociodemographic and clinical characteristics were also obtained from their medical records.Nine patients with a clinical diagnosis of Batten disease were identified. Genetic sequencing determined the presence of the previously described Costa Rican homozygous mutation in 8 of 9 cases. One patient did not have mutations in the CLN6 gene. In all cases where the Costa Rican CLN6 mutation was present, it was accompanied by a substitution in intron 2. Patients were born in 4 of the 7 Costa Rican provinces, with an average onset of symptoms close to 4 years of age. No parental consanguinity was present in pedigrees. Initial clinical manifestations varied between patients but generally included: gait disturbances, language problems, visual impairment, seizures and psychomotor regression. Cortical and cerebellar atrophy was a constant finding when neuroimaging was performed. Seizure medication was a common element of treatment regimens.This investigation supports that the previously characterized c.214G > T mutation is the most common causative NCL mutation in the Costa Rican population. This mutation is geographically widespread among Costa Rican NCL patients and yields a clinical presentation similar to that observed for CLN6 NCL patients in other geographies. CLN6 gene, which are responsible for variant late infantile or adult-onset forms (i.e. Kufs Disease Type A) of NCL [CLN6 gene is located on 15q23 and encodes a 311 amino acid transmembrane endoplasmic reticulum protein that functions in the ER to Golgi transfer of lysosomal enzymes [Neuronal ceroid lipofuscinoses (NCLs) constitute a group of genetic neurodegenerative diseases associated with motor and cognitive regression; progressive cortical, thalamic, and cerebellar atrophy; retinopathy; epilepsy; and a shortened lifespan , 2. NCLs) of NCL –6. The C enzymes . Downstr enzymes –12.The initial clinical manifestations of CLN6 disease generally begin between the ages of 2 and 4 years  and are CLN6 gene are observed mainly in patients of Portuguese, Indian, Pakistani or Czech descent, but have also been documented in countries such as Costa Rica, Sudan, Turkey and Japan [CLN6 gene mutation was defined [CLN6 gene  have also been reported [Mutations in the nd Japan , 21–23. nd Japan –26. From defined . This ge defined . Other preported . CurrentCLN6 gene mutation and any other mutations among Costa Rican NCL patients and to describe the main sociodemographic and clinical characteristics in patients diagnosed with NCL in the National Children's Hospital (HNN), the only pediatric hospital in Costa Rica, and part of the social security system (CCSS).Here, we aimed to determine the frequency of the c.214G>T CLN6 gene was performed for each study subject. Once the genetic results were obtained, the study participant´s parents received the genetic testing results with an explanation as part of their usual clinical visit.After obtaining approval from the Institution’s Scientific Ethics Committee (CEC-CENTRAL-CCSS) and authorization from the Director of Costa Rica´s National Children´s Hospital (HNN), the parents of all patients with a clinical diagnosis of Batten disease currently treated at the HNN Neurology Department were contacted, the study was explained and the investigators invited the parents so their child could participate. Subsequently, an appointment was scheduled in the HNN and, after explaining the study and obtaining informed consent (informed assent was not possible), the investigator collected a buccal swab for each participant. The samples were disassociated from any personal identification and transported in compliance with international regulations to the appointed laboratory. The genetic analysis of the samples was performed by Sanford Research  in two independent laboratories where DNA was extracted and next generation sequencing (NGS) for the The main sociodemographic and clinical characteristics from the study subjects were examined with a descriptive analysis based on existing medical records. These included: age, sex, place of birth and residence, age of initial symptom onset, age of clinical diagnosis, relevant medical history, main clinical manifestations and pertinent evaluations previously performed  at diagnosis, in addition to the result of the most recent neurological examination and current treatment.All study procedures were carried out in accordance with the Declaration of Helsinki, Good Clinical Practice Standards and local Costa Rican regulations.Nine patients with a previous clinical diagnosis of NCL were identified. The average age of the subjects when the DNA sample was obtained was 9.9 ± 2.7 years (mean ± SD). The initial symptoms appeared at 3.9 ± 0.6 years on average (range 3-5 years) and the typical age at the time of the clinical diagnosis was approximately 7 years (range 3-10 years). Females outnumbered males in a 2:1 ratio. Most patients  did not report a significant medical history before the onset of symptoms related to the disease.The Costa Rican mutation (homozygous nonsense variant in exon 3 c.214G> T in the CNL6 gene) was reported in approximately 90% (n = 8) of study participants. In all cases where the Costa Rican CLN6 mutation was present, it was accompanied by a substitution in intron 2 (c.198 + 104T>C, Table Most of the patients were born in and currently live in areas distant from San José, Costa Rica´s capital city. There was a fraternal relationship identified between two patients who live in the province of Limón. Nevertheless, there was no evidence suggesting the presence of specific geographic areas with a higher frequency of cases Fig. . LikewisGait disorders, vision loss and speech disturbances were the most frequent initial manifestations registered in medical records. Patient´s specific neurological signs and symptoms can be observed in Tables The review of subject´s medical records allowed for a qualitative comparison between the initial clinical manifestations and the findings from the most recent neurological examination, showing a substantial deterioration in physical and functional capabilities over the observation period Table . ValproiIn study patient A, genetic sequencing did not identify the Costa Rican mutation to confirm variant late infantile NCL. Full mutation analysis including: CLN1/PPT1, CLN2/TPP1, CLN3, CLN5, CLN6, CLN7, CLN8 and CLN10/CTSD was suggested for this patient. However, currently our Institution does not have the possibility to extend further genetic testing. After receiving the negative results for this patient new tests were performed and no data supporting alterations in metabolic or biochemical parameters was identified. Genetic confirmation of progressive leukoencephalopathy was also recommended. Currently the patient is receiving palliative care.Worldwide, it has been reported that the initial clinical manifestations in CLN6 disease occur between 2 and 4 years of age , howeverThe genetic alterations identified in the study population, pointedly the Costa Rican mutation and the substitution polymorphism in intron 2, have been previously reported , 22. ThiThe main signs and symptoms observed in study participants generally agree with what has been reported worldwide for CLN6 patients, where visual loss, the presence of rapid and involuntary muscle movements, seizures, ataxia, and mental and motor deterioration are the most frequent findings described , 17. SpeWhere neuroimaging studies were available, cortical and / or subcortical atrophy were common findings, as has been consistently reported in NCLs , 17–19. Current therapeutic options in this disease include symptom management for: seizures, sleep problems, extrapyramidal symptoms, behavior problems, anxiety and psychosis. Seizures in NCLs are often refractory to treatment and require the prescription of multiple antiseizure medications such as sodium valproate, lamotrigine, topiramate, levetiracetam, carbamazepine, and benzodiazepine derivatives , 38. TheRegarding the study participant in whom the c.214G>T Costa Rican mutation was not identified, it is possible that mutations in other NCL genes could be responsible for  the symptoms, as has been documented in other Costa Rican patients , 22. It Collectively, this investigation confirms the presence of the c.214G>T Costa Rican mutation in most Costa Rican patients with clinical symptoms of NCL and sustains that the clinical characteristics of these patients are similar to those described in other regions globally.


This study focused on the taxonomic and functional profiles of gut microbiomes according to the prevalence and incidence of nephrolithiasis. Stool samples from 915 Korean adults were collected at baseline. Participants were followed for a median of 4.0 years. We evaluated the biodiversity of the gut microbiota and taxonomic profiles associated with nephrolithiasis status, using 16S rRNA gene sequencing. Nephrolithiasis status was categorized into three groups: control (no-stone at both baseline and follow-up visits), incidental nephrolithiasis, and prevalent nephrolithiasis. Compared to the control and incidental nephrolithiasis, the prevalent nephrolithiasis showed a reduced evenness in alpha diversity. Nephrolithiasis was associated with a reduced abundance of some key taxa involved in short-chain fatty acid production. Moreover, the abundance of Bifidobacterium, which possess oxalate-degrading ability, was higher in the control. Conversely, there was no significant difference in the bacterial composition between the incidental and prevalent nephrolithiasis. In our study with repeated nephrolithiasis measurements, prevalent renal stones were associated with an altered gut microbiota composition compared to the control. Besides the known oxalate degradation pathway, other functional pathways inferred in this study require further investigation.The role of the gut microbiome in the development of renal stone diseases has not been well characterized Supersaturation of urine by constituents such as calcium, oxalate, or uric acid is the most widely accepted mechanism of nephrolithiasis5. However, its pathogenesis and risk factors are not yet fully understood.Nephrolithiasis, also known as kidney stone disease, is a common disease worldwide, with a prevalence of 5–10%. With a continuous increase in its prevalence and incidence, nephrolithiasis poses a considerable global health and economic burden8. However, research on this topic has mainly focused on Oxalobacter formigenes that utilizes oxalate as a principal energy substrate9. Its existence in feces has been correlated with a diminished renal excretion of oxalate and a decreased risk of stone recurrence7. Multiple previous studies have failed to identify a significant role of O. formigenes in nephrolithiasis, because isolation of O. formigenes from feces is not feasible9. However, recent analytical methods, such as 16S ribosomal RNA gene sequencing and shotgun metagenomics, have facilitated delineation of the gastrointestinal microbiome. At present, only five human studies, which have shown inconsistent findings, have evaluated the association between nephrolithiasis and the gastrointestinal microbiome14. All five of these studies included patients with symptomatic renal stone disease and were limited by small sample sizes and cross-sectional associations with single-time measurements.Recently, contribution of the gut bacteria to the pathophysiological gastrointestinal-renal axis has received increasing attention. Gastrointestinal microbiota has been suggested to play a role in oxalate metabolism, one of major proportion of nephrolithiasis, possibly by affecting the intestinal absorption of oxalate and renal stone formationThe present study aimed to examine the association between the prevalence and incidence of renal stones and the gut microbiota in a relatively large-scale study of 915 participants.15 between June 2014 and September 2014. A total of 1463 Korean adults aged 23 to 78 years who agreed to participate in this study and provided stool samples at Kangbuk Samsung Hospital Healthcare Screening Center16 were included. Fecal samples were obtained only at baseline. Abdominal ultrasonography is a routine part of a health checkup program.This prospective cohort study enrolled participants who underwent comprehensive annual or biennial examinations at the Kangbuk Samsung Hospital Healthcare Screening Center in South Korean = 7); use of antibiotics within 6 weeks prior to enrollment or probiotics within 4 weeks prior to enrollment (n = 72); history of cancer (n = 52); current use of antacid medication (n = 32); use of diabetes medication (n = 53); history of liver cirrhosis or identification of liver cirrhosis on an ultrasonography (n = 3); gout (n = 24); chronic kidney disease (n = 9); and samples with less than 5000 sequences (see below for further details) (n = 90). As the changes in the renal stone status over time were the focus of this study, participants with no follow-up visits by December 31, 2017, were also excluded (n = 302). None of the patients had a history of stomach, colon, kidney, or bladder surgery. Some individuals met more than one of the exclusion criteria. In the final analysis, a total of 915 participants were included. The characteristics of the study participants and the selection process are provided in detail in the Supplementary Methods. Dietary consumption was assessed using a 103-item self-administered food frequency questionnaire (FFQ) developed for use in Korea17. This validated FFQ was designed to measure usual food consumption during the previous year. Data were collected during the health checkup.Participants who met any of the exclusion criteria were not included in the analysis Fig. . We exclThe present prospective cohort study was conducted according to a protocol approved by the Institutional Review Board of Kangbuk Samsung Hospital (KBSMB 2013-01-245-12 and 2020-09-003). Written informed consent was obtained from all participants included in this study. Demographic, laboratory, and other clinical data, along with stool samples, were collected according to the ethical guidelines compliant with the Declaration of Helsinki.18. Based on the renal stone status at the initial and subsequent visits, patients were categorized into the following groups: G0, no renal stones (control), those without renal stones at the initial and subsequent visits; G1, incidental renal stones, those without renal stones initially but with renal stones at the follow-up visits; and G2, prevalent renal stones, those with renal stones at the beginning of the experiment. The median follow-up period was 4.0 years ; each participant was followed from baseline until the development of renal stones or the last follow-up examination, whichever came first.Abdominal ultrasonography was performed during the first and follow-up visits by experienced radiologists who were blinded to the study aim using a Logic Q700 MR with a 3.5 MHz transducer . All participants underwent abdominal ultrasonography in the supine or right-lateral position for both kidneys. Nephrolithiasis was diagnosed when hyperechoic structures causing acoustic shadowing were observed in the collecting system on ultrasonographyFresh fecal samples were collected from each volunteer, in a sterile container, immediately frozen at –20 °C, and stored at –70 °C within 24 h until further manipulation. Total DNA was extracted from the stool samples within 1 month of storage using the MOBio PowerSoil DNA Isolation Kit  according to the manufacturer’s instructions.19.To amplify and sequence the V3–V4 hypervariable region of the 16S rRNA gene, specific fusion primers were used . Libraries were pooled for sequencing, using the full complement of Nextera XT indices, and sequenced on the Illumina MiSeq platform  according to the manufacturer’s instructionshttps://qiime2.org )20 and the low-quality regions of the sequences and chimeras were removed. After denoising with DADA2, amplicon sequence variants (ASVs) were generated and regarded as 100% operational taxonomic units. Then, we constructed a feature table containing the counts of each unique sequence in each sample. We filtered out features present in only one sample, based on the suspicion that they did not represent actual biological diversity, and were attributed to polymerase chain reaction or sequencing errors.Demultiplexed sequences were processed using DADA2, which is a plugin of the QIIME2 package  was calculated using the UniFrac distance22 to estimate dissimilarities among group members by incorporating the phylogenetic distances between ASVs. Unweighted and weighted UniFrac distances were calculated to determine the presence or absence and abundance of ASVs, respectively. Non-phylogenetic β-diversity indices, such as the Bray–Curtis dissimilarity index23 were also used for the abundance data. We used pairwise permutational multivariate analysis of variance with 999 random permutations to test the significance of the differences among groups. The microbial community composition was depicted using a principal coordinate analysis plot. All tests were corrected for multiple tests. Basic statistical analyses were performed using R Studio , and microbial diversity plots were plotted using the “ggplot2” R package (version 3.3.2).The biodiversity of the samples (⍺-diversity) was calculated using the number of ASVs observed in each sample, the Shannon index, which accounts for both evenness and richness, Faith’s phylogenetic diversityhttps://qiime2.org). To investigate any significant differences in the relative taxa abundances from the phylum to genus levels among groups, we used two statistical tools from R (version 4.0.2): analysis of composition of microbiomes 24 and generalized linear models implemented in multivariate association with linear models (MaAsLin2). After adjusting for age, sex and BMI, we compared the abundance of taxa among G0, G1, and G2 in a pairwise manner. ANCOM compares the relative abundance of taxa among groups by the log-ratio of the abundance of each taxon to that of the remaining taxa, one at a time. The final significance was expressed in the empirical distribution of W at each taxonomic level. We used the taxa-wise false discovery rate (FDR) option with the significance level set to FDR < 0.05 to generate W statistics and a threshold of 0.6 for declaring a significant association. For the MaAsLin2 models, G0 was set as the reference group and compared with G1 and G2. The coefficient values and p-values that passed the significance threshold  are represented in the MaAsLin2 analyses. Linear discriminant analysis effect size (LEfSe) was used to detect potential nephrolithiasis-specific bacterial markers25. LEfSE utilizes a two-step statistical analysis including the nonparametric Kruskal–Wallis sum-rank test to identify bacterial taxa, which differ significantly in abundance between groups. This is followed by linear discriminant analysis to estimate the effect size of each differentially abundant feature. Only taxa with a linear discriminant analysis score (log10) greater than three (p < 0.05) were considered significantly enriched.Taxonomy was assigned to ASVs using a pre-trained naïve Bayes classifier and the q2-feature-classifier plugin against the Silva database (version r138.1) of the 16S rRNA sequence database in the QIIME2 package  numbers . We generated a metabolic pathway database (MetaCyc) using pathway abundance predictions from the EC-based gene family predictions27. We also inferred Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways levels by using the last publicly available mapping of KEGG orthologs to pathways (which is from around 2011)29. The predicted functional pathways were compared among the groups using statistical analysis of taxonomic and functional profiles (STAMP) version 2.1.330. Statistical differences in the pathways were tested using Welch’s t-test with a Benjamini–Hochberg FDR correction (q < 0.05) to adjust for multiple tests.For functional inferences of the microbial community, we conducted a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) (v2.2.0-b)Baseline characteristics of the enrolled participants according to renal stone status are shown in Table q = 0.041) and G1 (q = 0.041) . We found a significant decrease in the microbial evenness in G2 compared with that in G0  coefficient = 0.74; p = 0.01], the classes Actinobacteria  and Coriobacteriia , the order Bifidobacteriales , and the family Bifidobacteriaceae  compared to G0. According to the ANCOM results (W = 14), the class Fusobacteriia was more abundant in G2 than in G0. In addition, the MaAsLin results showed that Fusobacteriia was more abundant in G1  and G2  than in G0. At the genus level, the relative abundances of Dorea, Incertae sedis, Bifidobacterium, uncultured genus belonging to Lachnospiraceae, and Faecalibacterium were reduced by 10% (coefficient = –0.11) to 22% (coefficient = –0.25) in G1 compared to those in G0. We also found that the abundances of the genera Eubacterium eligens group, Dialister, Phascolarctobacterium, and Erysipelatoclostridium differed significantly between G0 and G2 (W > 170). The Eubacterium eligens group and Dialister were less abundant, while Phascolarctobacterium was more abundant in G2 than in G0. We observed that there were no significant differences in the composition of gut microbiota between G1 and G2. Notably, compared to G0, there were no shared significant taxa between G1 and G2, except for the class Fusobacteriia, in the MaAslin results. Figure We first used ANCOM, which has a low false-positive rate and allows covariate adjustmentts Table . After a25. Comparisons of G0 vs. G1 and G0 vs. G2 identified 10 and 11 taxa with linear discriminant analysis scores of > 3, respectively  and suggestive (FDR q < 0.25) metabolic pathways and how they contributed differently to each of the three groups according to renal stone status. Among the pathways, a MetaCyc pathway related to allantoin degradation IV (anaerobic) and three KEGG pathways related to styrene degradation, cyanoamino acid metabolism, and proteasome exhibited significant differences between G0 and G1, with decreased pathways in the G1 compared to those in G0  than O. formigenes (98%)33. Tavasoli et al. suggested that although some Lactobacillus and Bifidobacterium strains have oxalate-degrading potential, they may not be among the major oxalate-degrading bacteria of the gut microbiome. Recently, Denburg et al. reported that oxalate-degrading bacterial taxa, including Eggerthella lenta and several Lactobacillus species, were decreased in kidney stone formers34.Despite the essential role of Dorea, Incertae sedis, and Faecalibacterium were lower in incidental stone group than in no stone group. Additionally, compared with no stone group, the incidental stone group showed high abundances of Fusobacteria, Phascolarctobacterium, and Erysipelatoclostridium, and lower abundances of Eubacterium eligens group and Dialister. The association between these taxa and kidney stones has been reported in previous studies14. These observations imply that the gastrointestinal-renal axis may not solely rely on a single player or a limited number of species14. Ticinesi et al.14 suggested that oxalate-degrading activity may be shared by various taxa, influencing each other in a complex metabolic network.We also found that the abundances of Faecalibacterium in incidental stone group than in no-stone group, while there was no difference in abundances between prevalent stone and no-stone groups. A notable reduction in Faecalibacterium prausnitzii abundance has been reported in kidney stone groups in previous studies35. This bacterium is a principal manufacturer of short-chain fatty acids (SCFAs) within the human intestinal microbiome14. Recently, Liu et al.36 reported on the relationship between the gut microbiota and SCFAs regarding nephrolithiasis. They found that the abundance of pathways involved in SCFA production was positively correlated with the level of bacteria in the gut microbiota in the no-stone control group, but not in groups with kidney stones, which indicated that the gut microbiota of no-stone controls may tend to produce more SCFAs than that of nephrolithiasis patients. Moreover, the proportion of renal crystals was reduced after the administration of acetate, propionate, or butyrate in a rat model, which indicated that SCFAs could effectively prevent the formation of renal calcium oxalate crystals36. On the other hand, Dialister, which is also an SCFA-producing bacterium, was only depleted in prevalent stone group. We observed that there were no bacteria that showed significant differences in both the incidental stone and prevalent stone groups, when compared to no-stone group, and were associated with either incidental stone or prevalent stone group. This may be due to differences in the lifestyles of the incidental stone and prevalent stone groups. It is possible that patients with prevalent renal stones can alter their lifestyle behaviors, which may affect the gut microbiota.Interestingly, we observed a lower abundance of 37. These authors placed O. formigenes at the heart of a bacterial complex that was engaged in the breakdown of oxalate and avoidance of hyperoxaluria. In contrast to the more straightforward axial theory, which emphasizes the role of O. formigenes alone, these authors postulated the involvement of additional taxa that had oxalate-metabolizing properties, together with taxa that were triggered by the presence of oxalate and worked synergistically to break down salt, thus diminishing oxalate absorption.The potential gastrointestinal-renal axis in patients with nephrolithiasis was recently proposed by Ticinesi et al.38. Regarding styrene degradation based on microbiological activity, the citrate cycle can be affected39. Indeed, hypocitraturia or low urinary citrate excretion is a common feature in patients with nephrolithiasis. The pathway involved in the proteasome includes an interaction between calcium oxalate crystals and renal tubular epithelial cells40. Furthermore, the metabolism of xenobiotics by cytochrome P450 can be affected by the human gut microbiome, contributing to various diseases, including kidney stones41.Interestingly, the 16S data suggested that several metabolic pathways were related to renal stone formation. As allantoin is the final covert material created by uricase, allantoin degradation could be related to solubility. Therefore, allantoin degradation could increase uric acid contents42. Even though ultrasonography can be both appropriate and feasible in a large population study without radiation hazard, we cannot exclude misclassification of nephrolithiasis determined using ultrasonography. Furthermore, seasonal variations were not considered in this study. It is possible that participants in the incidental stone group had prior stone episodes but no stones on the baseline ultrasonography during the same season. Moreover, among the prevalent stone patients (n = 86), 76 (88.4%) had at least one follow-up visit, 50 (65.8%) were diagnosed with nephrolithiasis, and 26 (34.2%) had no stones detected at follow-up. Those with prevalent nephrolithiasis without stones at follow-up may represent a false positive result for nephrolithiasis or stone passage during follow-up. Unfortunately, detailed information on stone treatment or stone passage was not available, limiting our ability to differentiate between the two conditions. Additionally, subjects who had passed stones spontaneously and had no stones visible on ultrasound might have been misclassified as no stone group. Thus, no differentiation between symptomatic and asymptomatic stones and misclassification of stone formers might limit the implication of our findings. Another limitation of our study is that we only obtained the baseline fecal samples of participants without microbiome data at follow-up, which restricted our ability to examine the relationship between changes in the fecal microbiome and renal stone status over time. Further analysis including serial data of gut microbiota during follow-up periods is required to elucidate the longitudinal association between microbiome and renal stone changes over time. Although diet is a key factor in shaping the gut microbiota, the gut microbiome profile in individuals susceptible to renal calculi does not appear to be affected by diet in our study. Ticinesi et al.14 reported that dietary habits are apparently not involved in the nephrolithiasis-associated abnormalities of the gut microbiota composition, with the only possible exception being calcium intake. Moreover, additional elements may alter the constitution of the bacteria, which in turn may have a bearing on the pathophysiology of renal stone disease. Finally, our study was based on 16S rRNA gene sequencing, which provides limited information about bacterial genes and their functions. Whole metagenomic sequencing could expand our understanding of the strains associated with renal stones, their genes and functions, and metabolic pathways. Additionally, concordant analysis including the urinary and fecal microbiome could explain the relationship between the gut microbiome and nephrolithiasis more clearly44. Additional studies are required to discern the way in which the composition and purpose of the microbiome may impact therapy outcomes and influence renal stone disorders.This study incorporated a large population and investigated the association between the gastrointestinal microbiome and nephrolithiasis. However, the relevance of these results to current or potential clinical practices is not immediately apparent. In view of the multifaceted nature of the gut microbiome, further research is necessary to demonstrate the effectiveness of interventions aimed at restricting the absorption of oxalate across the intestinal barrier. Our study did not incorporate 24 h urine analysis or stone chemical analysis. Information on stone size and specific stone type, determined by detailed image or composition analyses, was also unavailable. We used abdominal ultrasonography to diagnose nephrolithiasis, although unenhanced computed tomography (CT) is considered a more accurate reference method. A recent study comparing ultrasonography and CT reported a fair sensitivity of 70.0% and specificity of 94.4% for detecting nephrolithiasisCompared with healthy controls, the incidental and prevalent renal stone groups exhibited an altered gut microbiota composition that could contribute towards nephrolithiasis physiopathology. Besides the known oxalate degradation pathway, other functional pathways inferred from taxonomic profiles have been suggested, which require further investigation in the future.Supplementary Information.