Flavobacterium, and Limnohabitans. The rich core community and low random elements compared to other relatively small cold lakes can be attributed to its simple hydrological network in a poorly-vegetated catchment, the long water-residence time (ca. 4 years), and the long ice-cover duration; features common to many headwater deep high-mountain lakes.Due to global warming, shorter ice cover duration might drastically affect the ecology of lakes currently undergoing seasonal surface freezing. High-mountain lakes show snow-rich ice covers that determine contrasting conditions between ice-off and ice-on periods. We characterized the bacterioplankton seasonality in a deep high-mountain lake ice-covered for half a year. The lake shows a rich core bacterioplankton community consisting of three components: (i) an assemblage stable throughout the year, dominated by Actinobacteria, resistant to all environmental conditions; (ii) an ice-on-resilient assemblage dominating during the ice-covered period, which is more diverse than the other components and includes a high abundance of Verrucomicrobia; the deep hypolimnion constitutes a refuge for many of the typical under-ice taxa, many of which recover quickly during autumn mixing; and (iii) an ice-off-resilient assemblage, which members peak in summer in epilimnetic waters when the rest decline, characterized by a dominance of  Declining snow and ice cover duration on lakes is expected with increasing climate warming, which can change ecological dynamics in ways that are hard to predict . High-moca. 2 ha lake size (or 20-m depth). In shallow lakes, light reaches the bottom throughout the lake; it can be said that the littoral extends to the whole lake, and water residence time is short (<1 year); the water column community can be “washout” during thaw and spring mixing. Most bacterioplankton studies on high-mountain lakes have been carried out in these shallow-type lakes. Less is known about deep lakes with a long water residence time and a profound hypolimnion with physical and chemical characteristics similar to those of the ice-cover period, a “habitat” lacking in shallow lakes. Therefore, we expected deep lakes to show rich bacterioplankton core communities responding to a higher hydrological stability, although in fluctuating environmental conditions throughout the year. Indeed, we hypothesized that the contrasting environmental conditions between the ice-on and ice-off periods should foster overall bacterial diversity by facilitating bacterial coexistence.Studies of a large number of lakes over hydrological networks have shown that bacterial communities inhabiting lakes are composed of core taxa, whose composition responds to in-lake environmental conditions, and a large fraction of mostly rare taxa, whose presence appears to be locally random and related to the characteristics of the downstream transport in the hydrologic network rather than lake conditions . In an aThe bacterial core community can be seen as a stable non-equilibrium coexistence of many taxa in a fluctuating environment , in whicBased on this hypothesis, we performed a detailed water column profile 1-year survey of the bacterioplankton community of a deep high-mountain lake (ice-covered for about 6 months per year). We used 16S RNA gene sequencing to characterize the bacterial communities and evaluated their spatial  and seasonal fluctuations. To discern between taxa showing seasonality and taxa with non-significant fluctuations across the year, we applied a procedure in which the partition of the whole set of samples in classes depicting the seasonality emerges from the patterns of change in the bacterial community , that iso 38′ 33″ N, 0o 36′ 13″ E), with an area of 0.24 km2, maximum and mean depth of 73 and 32 m, respectively, and water residence time of about 4 years. The lake is covered by ice and snow for 6–7 months  and relative surface irradiance (Iz) at depth (z) by assuming an extinction coefficient k = 1.7/zsd . During the ice-free season, water transparency was estimated with a Secchi disk . Total dissolved phosphorus (TDP), soluble reactive phosphorus (SRP), ammonium (NH4+), nitrite (NO2−), and dissolved reactive silica (DRSi) were determined by colorimetry using a segmented flow autoanalyzer . For TDP and SRP, the method (Bran+Luebbe G-175-96) was based on 4+ was determined by the blue indophenol method (B+L G-171-96) and NO2− by the Griess reaction (B+L G-173-96). DRSi was determined by molybdic-silicate reduction to heteropoly blue (B+L G-171-96). Nitrate (NO3−) and sulfate (SO42−) were measured by capillary electrophoresis . Dissolved inorganic nitrogen (DIN) was calculated as the sum of NO3−, NO2−, and NH4+. Total nitrogen (TN) was determined as nitrate after sample autoclave oxidation adding an alkaline sodium persulfate solution containing boric acid and sodium hydroxide  were determined using an elemental analyzer  and particulate phosphorus (PP) by acid persulfate wet oxidation followed by SRP analysis. Chla was extracted in 90% acetone with an ultrasonic homogenizer ; extracts were centrifuged  and filtered through a Whatman Anodisc 25 (0.1 μm), and quantified by spectrophotometry (Water samples were transferred to the laboratory in bottles without air space to avoid gas exchange that could modify the ydroxide . Dissolvydroxide . Dissolvotometry .® Green Supermix (Bio-Rad Laboratories), and the primers (0.5 μmol L−1). Standard curves within the range were obtained by serial dilutions of linearized plasmids containing a cloned fragment of the 16S rRNA gene.DNA was extracted using the phenol/chloroform method  without −1 BSA. Thermal cycling consisted of 25 cycles using the Applied Biosystems 2720 thermal cycler  for each sample. The duplicate products were pooled and purified following the Agencourt AMPure XP method, quantified using a Qubit fluorimeter (Thermo Fisher Scientific Inc.), and equimolarly pooled into the library pool. The product was inspected on 1.0% SB gel before Illumina Miseq (2 × ≥250 bp) sequencing at Microsynth (Switzerland).The diversity, structure, and composition of the bacterial communities were determined by sequencing the V3–V4 hypervariable region of the 16S rRNA gene. Amplicon libraries for each sample were generated using a two-step Polymerase Chain Reaction (PCR) protocol . The firl cycler . Replicade novo approach and the ‘gold’ reference database implemented in VSEARCH. For the OTU table construction, clusters with less than five reads were removed, and relative abundances were calculated by mapping back the quality-filtered reads at 97% identity. The OTU table comprised 749 OTUs that recruited 3,956,971 quality-filtered reads. Representative sequences from each OTU were classified using the SILVA (v.138) database  using the indicspecies R package  using the Hellinger distance , which r package . To obta package . Accordipost hoc pairwise comparisons with the HSD Tukey test  from March to June. The water column layers above 35-m depth were well-oxygenated throughout the year (>8 mg O2 L−1). At 60-m depth, the O2 concentration was slightly lower than in the upper layers during summer, autumn overturn, and early ice-covered stratification, but much lower during mid and late winter stratification, reaching hypoxia (<2 mg O2 L−1) from April to June.The Chla and O2 μg L−1 . Chla sp2 fluctuations. Regarding nutrients, phosphorus availability was low compared to nitrogen (−1) 500-fold higher than TDP (~0.02 μmol L−1). DOC levels were < 0.5 mg L−1. The particulate matter stoichiometry reflected the low P availability compared to carbon and nitrogen, particularly in the summer epilimnion. Despite the low water acid-neutralizing capacity, pH was usually stable and circumneutral (~6.5); pH < 6 only occurred in deep layers at the end of the ice-covered period, and pH > 7 in the Chla maximum during the ice-free period. The deviations were, respectively, related to CO2 accumulation and depletion. Sulfate and dissolved silica were always high compared to other nutrients.The chemical features were relatively stable in most of the water volume, as indicated by the similarity between the sample mean and median values . The strnitrogen , with av5 and 106 copies mL−1, with the highest levels detected in the hypolimnetic layers during summer (5 ± 1.3 × 105 copies mL−1). By contrast, the number peaked at 35-m depth in September and was also higher than average at 60-m depth during summer. The water column-integrated 16S rRNA gene copies peaked in September (~2 × 1013 copies m−2) during the highest Chla levels.Across the year, 16S rRNA genes ranged between 10g summer . From thThe identified 651 OTUs comprised 40 classes  and 147 Flavobacterium), autumn overturn , winter period , and thaw and deep summer layers .The community composition showed seasonal and depth patterns, although dissimilarities were more pronounced over time than between depths. The ordination indicated a continuous seasonal shift of the community composition rather than abrupt changes , with thThe clustering procedure resulted in an optimal partition of six seasonal clusters ,C, whichThe environmental conditions discriminating between seasonal bacterioplankton clusters were primarily related to those in the upper layers during summer , which d2, NH4+, and DRSi . Three groups were apparent , the most relevant families were Acidimicrobiia Ilumatobacteraceae, Actinobacteria Sporichthyaceae, Verrucomicrobiae , Gammaproteobacteria Comamonadaceae, and Bacteroidia Chitinophagaceae . Bactero, Limnohabitans), EP cluster indicator; OTU-8  ES indicator; and OTU-189 , TH indicator  included many under ice (UI) taxa but also many other OTUs not present in other samples, with functional traits different from the rest of the community . The basPolaromonas, one of the most abundant taxa in glaciers (The thawing period and deep hypolimnion (cluster TH) favored Burkholderiales, an order of the Gammaproteobacteria formerly known as Betaproteobacteria, one of the most abundant and diverse lineages in freshwater ecosystems . They miglaciers , was alsLimnohabitans and Rhodoferax, two aerobic anoxygenic phototrophic bacteria (AAPs) typically associated with productive periods , and Transfer (CGL2016–80124-C2-1-P) and the Catalan Government GECA (2017 SGR 910).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Robot-assisted spine surgery aims to improve the accuracy of screw placement. We compared the accuracy and safety between a novel robot and free hand in thoracolumbar pedicle screw placement.Eighty patients scheduled to undergo robot-assisted (40 patients) and free-hand (40 patients) pedicle screw placement were included. The patients’ demographic characteristics, radiographic accuracy, and perioperative outcomes were compared. The accuracy of screw placement was based on cortical violation and screw deviation. Safety outcomes mainly included operative time, blood loss, revision, and complications.P < 0.001). The rate of clinically acceptable screw position (grades A and B) was also higher in the robot-assisted group than in the free-hand group . The robot-assisted group had significantly lower screw deviation than the free-hand group . There was no robot abandonment in the robot-assisted group. No revision was required in any of the groups.A total of 178 and 172 screws were placed in the robot-assisted and free-hand groups, respectively. The rate of perfect screw position (grade A) was higher in the robot-assisted group than in the free-hand group (91.0% vs. 75.6%; Robot-assisted pedicle screw placement is more accurate than free-hand placement. The second-generation TiRobot–assisted thoracolumbar pedicle screw placement is an accurate and safe procedure.Trial registration retrospectively registered Pedicle screw fixation is the most commonly used internal fixation method in thoracolumbar spine surgery , 2. It pTiRobot system, a multiple-indication surgical robot developed by Beijing Jishuitan Hospital and Beijing TINAVI Medical Technologies, has achieved good results in clinical orthopedic surgeries –15. In aThe purpose of this study was to compare the accuracy and safety between the second-generation TiRobot and free hand in thoracolumbar pedicle screw placement.This retrospective study recruited patients scheduled to undergo thoracolumbar pedicle screw placement in our hospital between December 2020 and June 2021. The study was approved by the Ethics Committee of Beijing Jishuitan Hospital (Number: 201911-05-01), and informed consent was obtained from all patients.The inclusion criteria were as follows: (1) scheduled for thoracolumbar pedicle screw placement; (2) age over 18 years; (3) willingness to participate in the study. The exclusion criteria were as follows: (1) previous surgical history of thoracolumbar spine; (2) active infection; (3) presence of scoliosis greater than 20°; (4) history of spinal tumors or tuberculosis.Two groups of patients undergoing thoracolumbar pedicle screw placement were retrospectively included. The patients were divided into the robot-assisted (RA) group and the free-hand (FH) group depends on whether the robot was used intraoperatively. RA or FH surgery was chosen by the patient after the surgeon clarified the details of both procedures. All thoracolumbar pedicle screw placements were performed by the same team of spine surgeons. Each surgeon performed the same amount of both surgeries. The RA group comprised patients (n = 40) consecutively treated under robot-assisted surgery. The FH group comprised patients (n = 40) consecutively treated using fluoroscopy-guided FH surgery.The robot-assisted procedures were performed using the second-generation TiRobot system . This robot inherits the precision advantage of the previous generation and makes the surgical procedure more efficient through the “intelligent surgical module” design Fig. . A newlyThe robot-assisted procedures were performed in accordance with the “guideline for thoracolumbar pedicle screw placement assisted by orthopedic surgical robot ” Fig. 22. The C-Cortical violation was evaluated on postoperative computed tomography (CT) by two doctors blinded to this study according to the Gertzbein and Robbins scale , as follScrew deviation was measured by comparing the actual guidewire positions to the planned guidewire positions  through merging the postoperative CT images with the planning images at the same depth in the axial and sagittal planes , 10, 13 Information about the operating time, intraoperative blood loss, complications, robot abandonment, and revision surgeries was recorded and compared.t test and Mann–Whitney U test (continuous variables) were used to evaluate statistical differences. In all of the statistical analyses, P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS software . The normality of distribution for continuous variables was verified using the Shapiro–Wilk test. The Chi-square test and Fisher’s exact test  or Student’s A total of 178 pedicle screws were placed in the RA group, while a total of 172 pedicle screws were placed in the FH group. There was no statistically significant difference in age, gender, and body mass index between the groups (Table P < 0.001). The rate of clinically acceptable screw position (grades A and B) was also higher in the RA group than in the FH group . The RA group had significantly lower screw deviation than the FH group   was higher in the RA group than in the FH group  mL vs. 300  mL, P = 0.439]. There was no robot abandonment in the RA group. No revision surgeries and screw-related complications were noted in any of the groups  min vs. 120 90, 135) min, 5 min, P The present study compared the accuracy and safety between RA and FH pedicle screw placement. The main finding is that the RA technique with an upgraded robot system was superior to the FH technique in terms of cortical violation and screw deviation.Previously, we investigated clinical application of the TiRobot orthopedic robot in cervical and thoracolumbar surgery in prospective randomized controlled studies. The results showed that the accuracy and safety of the robot in cervical spine internal fixation were better than those of the FH surgery, with the perfect screw position rate of 87.6% and a screw deviation of 0.83 mm . The accHowever, the use of TiRobot to perform pedicle screw placement has some shortcomings: (1) Before three-dimensional image scanning, an image registration calibrator needs to be installed, and fluoroscopy is required to ensure that the marking points on the calibrator are captured by the anteroposterior–lateral images at the same time; (2) A special individual is required to handle the main control station to control the robot; (3) The tracker in the end of the robotic arm has a single-plane design, leading to a limited tracking angle.Through the collaborative innovation by doctors and engineers, the second-generation TiRobot system was developed. Its main upgrades are as follows: (1) integration of the image registration calibrator with the C-arm, which reduces the difficulty of image registration to save operation time; (2) surgeons can directly click the touch screen in the main control station and the operating button in the robotic arm to fully control the robot; (3) the 360-degree omnidirectional tracer in the robotic arm is easier to be recognized by the optical tracking system, which reduces operation time of adjusting the position of optical tracking devices. A review of the literatures found that the clinical outcomes of second-generation TiRobot were similar to first-generation TiRobot.In our study, more screws had the perfect position and clinically acceptable position in the RA group than in the FH group. The higher accuracy achieved in the RA group is consistent with previous reports. Fatima et al.  performeAlthough one of the purposes of the improvements of the second-generation TiRobot is to shorten the operation time, this effect was not reflected in this study. This is our first 40 cases of second-generation TiRobot-assisted spine surgery. It is possible that the operation time was not shortened due to the early learning curve required by the surgeon to adapt to the new human–computer interface. Future studies with larger samples are needed to confirm whether the use of this robot can actually shorten the operation time.The present study had some inherent limitations. Firstly, this study was a retrospective analysis of radiological and perioperative outcomes. Follow-up assessments are needed to assess the long-term outcomes. A multicenter prospective study with a larger sample size is needed to verify the findings of this study. Secondly, this study did not directly compare the clinical outcomes of second-generation TiRobot with first-generation TiRobot, and further in-depth research is needed in the future.Robot-assisted pedicle screw placement is more accurate than FH placement. The second-generation TiRobot-assisted pedicle screw placement is an accurate and safe procedure in thoracolumbar spine surgery.


Mycobacterium tuberculosis complex (MTBC) has become one of the leading causes of death in humans and animals. Current research suggests that the transmission of MTBC in the environment indirectly transmit to humans and animals with subsequent impact on their wellbeing. Therefore, it is of great significance to take One Health approach for understanding the role of MTBC in not only the interfaces of humans and animals, but also environment, including soil, water, pasture, air, and dust, etc., in response to the MTBC infection. In this review, we present the evidence of MTBC transmission from environment, as well as detection and control strategies in this interface, seeking to provide academic leads for the global goal of End Tuberculosis Strategy under multidisciplinary and multisectoral collaborations.Tuberculosis caused by the  Mycobacterium tuberculosis complex (MTBC) constitutes a significantly genetically similar group of bacteria that cause tuberculosis in a wide range of hosts. They are rod-shaped, acid-base-fast, aerobic, slow-growing intracellular pathogens that destroy phagosomal cells to maintain and evade the immune system  and real-time PCR (RT-PCR) for rapid identification of MTBC targeting 16S rRNA, insert sequences IS6110 and IS1081, and genes encoding MTBC-specific proteins, such as MPB70 have been applied , 42. SevSpecific identification and genotyping of MTBC are considered important means for understanding transmission among humans, animals, and the environment. Various genotyping techniques have been developed to distinguish MTBC isolates from shared environment. These methods can further identify different MTBC strains and will enable characterize the origin, spread and mode of transmission of MTBC . The mosThe rapid development of whole-genome sequencing (WGS) including next-generation sequencing and long-read technology sequencing has made it possible to identify genetic information of MTBC in environmental samples. WGS can sequence complete genomes of multiple strains simultaneously, saving time, streamlining workflow, and provide more information than traditional methods . WGS resMinimizing the MTBC in the environment using One Health approach helps protect human and animal health. Establishing an effective disinfection system is the key to controlling MTBC in the environment. As for indoor environments such as hospitals and prisons, aerosols are still the major way to spread tuberculosis. The primary mean of air disinfection is natural ventilation which has the advantage of wide availability, low cost, and high efficacy. In 10 studies evaluating environmental interventions, including 31,776 human participants, mechanical ventilation reduced infections by 2.9 to 14% . HoweverIn pastures, especially cattle farms, there is high possibility of the contamination with feces, droplets, and nasal mucus. This untreated manure potentially increases the risk of indirect transmission of tuberculosis . TherefoRoutine disinfection is also essential to control the spread of MTBC from environment to humans and animals. A permanent disinfection channel should be set up at high-risk regions, particularly, such as the entrance of the farm, so that personnel, vehicles, and materials can be appropriately disinfected especially when entering and leaving the farm. Effective disinfectant, such as 20% lime milk, can be applied in the disinfection channel. To ensure the actual effect of the disinfectant, replacement is required every 15 days. The surrounding environment of the cowshed (including the sports field) shall be disinfected with 2% sodium hydroxide or sprinkled with quicklime once a week. Sewage tanks, cesspits, and sewer outlets shall be disinfected with bleaching powder once a month. All utensils in the barn should be disinfected regularly. The delivery room should be disinfected once a week besides immediate disinfection before and after delivery. Milking staff, milking machines and other tools must be cleaned and disinfected every time. Additionally, it should be noted that contamination of milk by disinfectants may occur during environmental disinfection in the milking parlor.The environment contamination by wild animals may also play an important role in tuberculosis epidemiology. It is believed transmission and infection occur through the shedding of infectious bacilli of animals, and this phenomenon is well-described in badgers, wild boars, and deer , 54, 55.Currently, human and animal tuberculosis control programs have been implemented under normal conditions. Under the long-term efforts of WHO, OIE, FAO and the European Union, the joint aim is to reduce the percentage of tuberculosis deaths by 95% and the incidence of tuberculosis by 90% by 2035 compared with 2015 . HoweverTherefore, One Health approach enhances the awareness of the prevention and control of MTBC in the environment, demonstrating an essential way to the goal of End Tuberculosis Strategy . On the At the national level, the government needs to initiate MTBC monitoring and prevention system in the environment. The health department should closely monitor the disinfection records of medical and health institutions. In addition to the harm of MTBC infection itself, in a high-risk environment contaminated by MTBC, HIV co-infection is the most important risk factor for the development of latent tuberculosis into active tuberculosis . In the For tuberculosis institutes and laboratories, it is necessary to strengthen scientific research on MTBC in the environment, develop molecular biology detection technologies, and improve the accuracy and timeliness of MTBC detection in the environment . EstabliThere is already substantial evidence of MTBC contamination in indoors, farms, and natural environments, with the potential to infect humans and animals. However, studies on tuberculosis transmission have mainly focused on direct transmission, and the potential impact of contamination of MTBC in the environment is frequently neglected. Additionally, the inappropariate detection and disinfection methods even prolong the persistence of MTBC in the environment. This review underscored the integration of environmental MTBC into tuberculosis control program, which relies on multidisciplinary and multisectoral collaborations. Also, it is of necessity for the endorsement and input of governments on surveillance, academic research, tuberculosis control products R&D, public awareness comprehensively to initiate the national and international programs at human-animal-environment interface by utilizing One Health approach to reduce the tuberculosis burden.HZ wrote the article. ML was responsible for finding references. WF and SS provided their detailed and constructive comments. XF revised the article. All authors contributed to the article and approved the submitted version.This work was supported by China Agriculture Research System (CARS-36).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


DL-glyceraldehyde as a substrate. Among the ginsenosides tested, ginsenoside Rh2, (20S) ginsenoside Rg3, (20R) ginsenoside Rg3, and ginsenoside Rh1 inhibited RLAR significantly, with IC50 values of 0.67, 1.25, 4.28, and 7.28 µM, respectively. Moreover, protopanaxadiol, protopanaxatriol, compound K, and ginsenoside Rh1 were potent inhibitors of HRAR, with IC50 values of 0.36, 1.43, 2.23, and 4.66 µM, respectively. The relationship of structure–activity exposed that the existence of hydroxyl groups, linkages, and their stereo-structure, as well as the sugar moieties of the ginsenoside skeleton, represented a significant role in the inhibition of HRAR and RLAR. Additional, various modes of ginsenoside inhibition and molecular docking simulation indicated negative binding energies. It was also indicated that it has a strong capacity and high affinity to bind the active sites of enzymes. Further, active ginsenosides suppressed sorbitol accumulation in rat lenses under high-glucose conditions, demonstrating their potential to prevent sorbitol accumulation ex vivo. The findings of the present study suggest the potential of ginsenoside derivatives for use in the development of therapeutic or preventive agents for diabetic complications.This present work is designed to evaluate the anti-diabetic potential of 22 ginsenosides via the inhibition against rat lens aldose reductase (RLAR), and human recombinant aldose reductase (HRAR), using  According to the World Health Organization, approximately 200 million people—5.9% of the adult population worldwide—suffer from diabetes, a very common disease that has assumed epidemic proportions. As a result, it is expected that by 2025, the disease will have a significant impact on human health ,2. DiabeP. notoginseng (Burk.), P. quinquefolius L, and P. ginseng (Meyer) are the three main commercial varieties )] was produced to determine the kinetic mechanisms of inhibition  against inhibitor concentration [I] used to determine the type of enzyme inhibition; it was used to identify the dissociation or inhibition constant (iK) for the enzyme-inhibitor complex. Dixon plots  for RLAR and HRAR inhibition were obtained in the presence of 100, 50, and 10 mM RLAR, and 20, 10, and 5 mM HRAR DL-glyceraldehyde substrate. Thus, the type of enzyme inhibition was determined by interpreting the Lineweaver-Burk plots, and the inhibition constants (iK) were determined by interpreting the Dixon plots. Lineweaver-Burk and Dixon plots [1/enzyme velocity  in TC-199 medium comprising 100 U/mL penicillin, 15% fetal bovine serum, and 0.1 mg/mL streptomycin. The ginsenosides were liquified in DMSO. The lenses were separated into three groups with a medium containing RLAR-active ginsenosides and 25 mM glucose. Each lens was placed in a medium (2.0 mL). The sorbitol was calculated by HPLC [Lenses were isolated from Sprague-Dawley rats through cultures with sterile conditions and an atmosphere  , and RLAp-value < 0.05 was considered statistically significant.All experimental results were expressed as the mean ± SEM of triplicate experiments. Statistically significant values were analyzed using analysis of variance (ANOVA) and Duncan’s test . A In summary, this study investigated the RLAR and HRAR inhibitory activities of ginsenoside derivatives. Ginsenosides demonstrated high inhibitory activities in two in vitro assays, and their potentially beneficial effects on diabetic complications make ginsenosides an excellent ingredient for functional foods. Additionally, the eight most active ginsenosides demonstrated potent inhibitory activities on sorbitol accumulation in isolated rat lenses. Our data indicated as well that aglycone PPD or PPT, and a hydroxyl group with a single sugar moiety containing ginsenosides, inhibit aldose reductase activity in vitro, and the mode of action depends upon the ginsenosides’ structure. These structure−function relations could be useful in designing new aldose reductase inhibitors based upon ginsenosides. Collectively, this study together with our previous finding on the AGE formation inhibitory activity, suggests that ginsenoside derivatives can be potent functional food ingredients as RLAR and AGE inhibitors and can be used as a naturotherapy for diabetic complications. Further investigation to clarify their beneficial/harmful effects in vivo are underway in our laboratory.


A detailed depiction of nutrient foramina is useful for defining guidelines and minimising iatrogenic damage during hip surgery. Therefore, this study aimed to define the location and frequency of nutrient foramina in the proximal femur using mapping techniques.One hundred dry human cadaveric proximal femurs, comprising 56 left and 44 right femurs, were scanned using a three-dimensional scanner, with scanning distance 200 mm, precision 0.01 mm, and measuring point 0.04 mm. The image resolution of 1,310,000 pixels was obtained. Digital imaging models were acquired from the proximal femur surface. All the nutrient foramina in each model were identified and marked. The nutrient foramina models were superimposed on one another and oriented to fit a standard template of the femur’s proximal aspect. Three-dimensional mapping in the proximal femur’s nutrient foramina was performed.The nutrient foramina’s location and dense zones were identified. The dense zones were distributed along the vascular course and gaps between the muscle attachment sites. Eighteen dense zones were identified and found to be location-dependent. They were located in the central part of the fovea capitis femoris, subcapital and basicervical areas of the femoral neck, and muscle attachment gaps of the femoral trochanter.The terminal branch of the nutrient vessels entering the nutrient foramina is at risk for iatrogenic damage during hip surgeries, especially in cases of close bone exposures. There are 18 dense zones that need to be considered for a safer approach to the proximal femur. To minimise iatrogenic damage to the nutrient vessels entering the nutrient foramina, the dense areas should be avoided when technically possible. The nutrient foramen is a principal entry pathway for nutrient vessels, which are necessary for fracture healing or integration of implanted prostheses. The blood supply to the femoral head (FH) is derived primarily from the lateral femoral circumflex artery (LFCA) and the medial femoral circumflex artery (MFCA), which is a major contributor , 2. The Previous studies have reported the location and number of nutrient foramina in various bones using macroscopic evaluation –10 and tThis study aimed to define the location and frequency of nutrient foramina in the proximal femur. We hypothesised that there are dense nutrient foramina zones in the proximal femur.N = 20); (2) the presence of the epiphyseal growth plate of the FH (N = 8); and (3) damaged proximal femur (N = 43). One hundred dry femur specimens comprising 56 left and 44 right femurs were included.A total of 171 unpaired dry specimens of adult femurs were obtained from the Department of Anatomy to study the nutrient foramina’s 3D location and distribution in the proximal femur. General information regarding these specimens was unknown. Specimens were excluded based on the following criteria: (1) significant osteoarthritis or morphological changes within the FH . The nutrient foramina were densely located in the specimens with the distribution of the nutrient foramina being approximately parallel to the course of the nutrient vessels. The nutrient foramina in the femoral trochanter regions were mainly located in gaps between the muscle attachment sites . The 18 dense zones (DZs) identified were location-dependent (3). See also Of 171 specimens, 71 were excluded. The nutrient foramina of the proximal femur of all the 100 specimens were visualised in a standard template . Meanwhile, there was a reduction in the density of nutrient foramina from the central to peripheral regions.The DZ was located in the centre of the fovea capitis femoris .Two DZs were distributed around regions of vessel bifurcation and channels through which the terminal branches of the anterior retinacular arteries enter the FH . The nutrient foramina cluster varied from about 1/2 to 1/7 of the femoral neck length, toward the FH, showing a gradually decreasing trend from the anterior to posterior regions . Two zones were located at the two sides of the basicervical femoral neck (3E).There were three DZs. The first zone was located in the subcapital area of the femur’s neck, where superior retinacular arteries insert into the FH (3E).The DZ was located, about 1/7 of the femoral neck length, in the half posterior regions of the transcervical femoral neck through which the inferior retinacular arteries entered the FH . The first zone was situated in the medial-inferior regions of the attachment site of the gluteus minimus (GM-1), lateral regions of the attachment site of the articular capsule (AC), and superior regions of the proximal attachment site of the vastus lateralis (VL). The second zone was located in the medial-inferior regions of the attachment site of the VL, lateral regions of the attachment site of the AC, and superior regions of the proximal attachment site of the vastus intermedius and vastus medialis (VM).There were two DZs . The DZ was located in the regions below the distal attachment site of the gluteus medius (GM-2). The first lower DZ was located between the anterior regions of the attachment site of the GM-2 and the posterior regions of the attachment site of the GM-1. The second lower DZ was located between the posterior and superior regions of the attachment site of the VL and the inferior regions of the attachment site of the GM-1 and GM-2.There was one DZ and two lower DZs (3D). The second zone was located in the trochanteric fossa’s central part .There were two lower DZs. The first zone was located in the anterior part of the lesser trochanter, which was predominately distributed among the inferior attachment site of the AC, anterior attachment site of the iliopsoas, and posterior attachment site of the VM . The DZ was located along the pertrochanteric crest and medial part of the attachment site of the quadratus femoris (QF). The first lower DZ was located below the attachment site of the GM-2 and above the attachment site of the QF. The second lower DZ was located in the lateral attachment site of the QF and above the proximal attachment site of the gluteus maximus. The third lower DZ was located at the proximal-medial attachment site of the adductor magnus and distal-lateral attachment site of the iliacus and pectineus.There was one DZ and three lower DZs (Our results showed that there were 18 dense nutrient foramina zones in the proximal femur, confirming our hypothesis. We used the nutrient foramina mapping technique inspired by Armitage et al.  to analyDetailed insights into the position of the nutrient foramina in the long bones can be useful in surgeries and medicolegal cases , 22. TheGreater awareness of the nutrient foramina’s morphology can significantly affect the successful planning of hip surgeries. There have been very few studies on the effects of the surgical approach in hip surgeries on the impairment of blood supply to the proximal femur through the nutrient foramina. Previously, distribution of the nutrient foramina around the FH and neck junction was roughly described using a clock system that was not accurate enough for use in the clinical setting . Herein,Previous studies on the blood supply of the proximal femur focused on the circulation of extracapsular blood vessels. However, the nutrient foramina as the entrance and exit of a bone’s blood vessel is also the last way to protect the blood supply of the FH during hip surgery. Therefore, dense nutrient foramina zones need attention. The choice of surgical approaches to the proximal femur depends on the surgical goal and surgeon preference –33. In aThere have been several studies on the blood supply to the hip aimed at improving the outcomes for hip procedures . HoweverTo our knowledge, this is the first study to elucidate the location of nutrient foramina of the proximal femur by utilising 3D mapping techniques based on 3D image data. The 3D mapping techniques have been widely used in studies to describe the morphology of various bone fractures , 16. TheThere were some limitations to this study. First, this was a descriptive macrostructural study on the map of nutrient foramina in the proximal femur; thus, the map of microstructures, such as trans-cortical vessels , needs tIn conclusion, the nutrient vessels entering the nutrient foramina are at risk of iatrogenic damage during hip surgery, especially when soft-tissue exposure is close to the course of the vessels and gaps between the muscle attachments. Eighteen DZs for iatrogenic vessel injury were identified: one was located in the central part of the fovea capitis femoris; six were located in the subcapital and basicervical areas of the femoral neck; and 11 were located in the muscle attachment gaps of the femoral trochanter. To minimise iatrogenic damage to nutrient vessels entering the nutrient foramina, the direction of soft-tissue exposure should be parallel to the course of vessels where technically possible.


Escherichia coli); human fecal-associated microbial source tracking (MST) markers (crAssphage and HF183/BacR287); and a suite of clinically relevant antibiotic resistance genes  and a gene associated with antibiotic resistance (intl1). Mean levels of FIB and clinically relevant ARGs (blaCMY and KPC) were similar across sites, while MST markers and intI1 occurred at higher mean levels at the natural site. The human-associated MST markers positively correlated with antibiotic resistant-associated genes at both sites, but no consistent associations were detected between culturable FIB and any molecular markers. For all microbial indicators, generalized additive mixed models were used to examine diurnal variability and whether this variability was associated with environmental factors . We found that FIB peaked during morning and early afternoon hours and were not associated with environmental factors. With the exception of HF183/BacR287 at the urban site, molecular MST markers and intI1 exhibited diurnal variability, and water temperature, pH, and turbidity were significantly associated with this variability. For blaCMY and KPC, diurnal variability was present but was not correlated with environmental factors. These results suggest that differences in land use  both adjacent and upstream may impact overall levels of microbial contamination. Monitoring agencies should consider matching sample collection times with peak levels of target microbial indicators, which would be in the morning or early afternoon for the fecal associated indicators. Measuring multiple microbial indicators can lead to clearer interpretations of human health risk associated with exposure to contaminated water.Water bodies that serve as sources of drinking or recreational water are routinely monitored for fecal indicator bacteria (FIB) by state and local agencies. Exceedances of monitoring thresholds set by those agencies signal likely elevated human health risk from exposure, but FIB give little information about the potential source of contamination. To improve our understanding of how within-day variation could impact monitoring data interpretation, we conducted a study at two sites along the Chattahoochee River that varied in their recreational usage and adjacent land-use , collecting samples every 30 min over one 24-h period. We assayed for three types of microbial indicators: FIB (total coliforms and  Escherichia coli, among other organisms, are commonly used by state and local environmental agencies as markers of elevated fecal contamination levels in water. FIB have been a critical tool for monitoring water bodies and minimizing health risk from water exposure through recreation, drinking, and agricultural irrigation , which include fecal coliforms, enterococci, and rigation . The Unirigation .State agencies predominantly culture FIB from 100 ml water samples and use these data to provide public health advisories to stakeholders  in addition to regulatory action. However, research has demonstrated that in many untreated surface water bodies, the levels of FIB poorly predict the risk of gastrointestinal illness  and do nFIB also display high spatial and temporal variation in marine and fresh waters . For exaGiven that human fecal contamination carries the greatest risk to human health , microbiBesides enteric viruses, bacteria, and other pathogens of concern, urban runoff and treated wastewater effluent can introduce antibiotic resistant organisms (AROs) and antibiotic resistance genes (ARGs) into receiving water bodies . Sewage Understanding how microbial indicators vary over space and time could improve our ability to assess human health risk, as the co-occurrence of elevated FIB, human-associated MST markers, and ARGs could signify a much higher exposure risk than the detection of any of these microbial indicators alone. Using FIB in combination with MST markers can improve water quality monitoring and assessment in marine  and fresE. coli levels at three major sites in the Chattahoochee River. Weekly monitoring from 2000 to 2008 showed that during stormflow conditions at the urban Atlanta location (Paces Ferry), levels of E. coli routinely exceeded USEPA recreational water quality criteria of 126 colony forming units (CFU) per 100 ml of water , human fecal-associated MST molecular markers (crAssphage and HF183/BacR287), and a suite of clinically relevant antibiotic resistance genes  and the mobile element class 1 integron gene associated with antibiotic resistance (intl1) [hereafter referred together as antibiotic resistance-associated genes (ARGs)]. Our primary objectives were to: (a) quantify overall levels of contamination between sites with distinct public water use and surrounding land uses; (b) examine correlations between FIB, MST markers, and ARGs; (c) determine the diurnal variability of FIB, MST markers, and ARGs between sites; and (d) evaluate the relationship between environmental factors and diurnal variability between sites.To gain a holistic assessment of exposures to microorganisms associated with negative health outcomes and to inform monitoring approaches in the Chattahoochee River, we conducted a high-resolution temporal study at two sites along the river that varied in their recreational usage and surrounding land use , collecting samples every 30 min over one 24-h period. We assayed samples for culturable FIB  and urban (Paces Ferry) site along the Chattahoochee River . Cochran2) were extracted from the National Solar Radiation DatabaseWater samples were collected at the shoreline of each site in 30-min intervals for 24 h from 04:00 on April 9, 2016 to 03:30 on April 10, 2016 for a total of 48 samples per site. At each interval, ~1 L of water was collected in a Whirl-pak bag  at ankle-deep depth and placed on ice. Water temperature and pH were recorded at each sampling event with a WaterproofPocket pH Test . Water turbidity was also measured on-site using a 2100Q Portable Turbidimeter . Because sunlight is known to impact the environmental persistence of FIB and MST markers, Global Horizontal Irradiance (GHI) measurements (W/mE. coli per 100 ml (Samples were processed within 5 h of collection using the Colilert most probable number (MPN) method with IDEXX QuantiTray/2000 assays  to enumerate total coliforms and r 100 ml . The ranFor molecular assays, 250 ml of each sample were filtered through 0.45 μm-pore size nitrocellulose filters  and stored at −80°C until extraction. DNA was extracted from filters using the Qiagen DNeasy PowerWater extraction kit  according to manufacturer instructions. Briefly, filters were subject to bead beating lysis and processed with a final nucleic acid elution volume of 100 μl. All extracts were stored at −80°C until qPCR assays were performed.intI1; blaCTX-M, blaCMY; MCR, KPC, VIM, NDM; Two MST markers, crAssphage target 056  and HF18Briefly, each sample was analyzed by qPCR in triplicate using 2 μl of extract in 50-μl reactions using TaqPath™ qPCR Master Mix  for detection of the AR-associated genes due to the necessity for high purity, ARG-free polymerase. For the MST markers, 2 μl of extract in 25-μl reactions using Environmental MasterMix 2.0  on an Applied Biosystems (ABI) 7,500 thermocycler . The thermal cycling parameters for all qPCR assays were as follows: 10 min denaturation at 95°C and then 45 cycles of 95°C for 15 s and 60°C for 1 min. To test for amplification inhibition, the HF183/BacR287 qPCR was run with an internal amplification control , and inh0–105 copies per reaction; A linearized synthetic DNA plasmid was used as a reference standard  to enumerate the genetic markers consisting of six 10-fold serial dilutions (10Extraction volume refers to the volume of water that was membrane filtered (250 ml), template volume refers to the amount of template used in each well (2 μl), gene copies per reaction refers to the raw quantity from the qPCR assay, and volume processed refers to the volume of template after DNA extraction (100 μl). We standardized concentrations as gene copies per 100 ml of water.10 means of FIB levels over the entire study period were compared between sites using two-sided t-tests. For crAssphage, HF183/BacR287, blaCMY, intI1, and KPC the mean concentration per triplicate qPCR was calculated, and then the log10 mean for each target was calculated over the entire study period and compared between sites using a two-sided t-test. In cases where the variance was unequal , a Welch’s t-test was conducted instead. Non-detected samples were excluded from two-sided and Welch’s t-tests to avoid artificially minimizing the standard error. Next, associations between FIB and MST markers, FIB and ARGs, and MST markers and ARGs at each site independent of environmental factors  were evaluated using Kendall’s non-parametric measure of correlation. Here, non-detected samples were set to half the LOD for each qPCR assay so the analysis could account for tied ranks.Statistical analyses were conducted using R 4.1.0 . The logE. coli, crAssphage, HF183/BacR287, blaCMY, intI1, and KPC was evaluated using Generalized Additive Mixed Models (GAMMs). Specifically, GAMMs were used to determine if the measured microbial indicators exhibited significant diurnal variability and how strongly environmental factors  were associated with this variability. GAMMs facilitate inferences about nonlinear dynamics over time through the incorporation of smooth functions as predictors  using the gamm function in the mgcv package , E. coli , blaCMY , and KPC  mean concentrations between the natural and urban sites , HF183/BacR287 , and intI1  were significantly higher at the natural site compared to the downstream urban site . Escherichia coli had moderately positive correlations with all MST markers and ARGs (τ range: 0.31–0.57). CrAssphage and HF183/BacR287 most strongly positively correlated with intI1 (τ = 0.53) and moderately correlated with blaCMY and KPC . Similar to trends at the natural site, crAssphage and HF183/BacR287 most strongly correlated with intI1 (τ range: 0.55–0.59) and also moderately correlated with blaCMY and KPC (τ range: 0.41–47).At the urban site, total coliforms did not correlate with MST markers or ARGs. E. coli were consistently detected and showed similar nonlinear, temporal trends at both sites , and there was no significant difference in the diurnal variability trend at the natural site  but did at the natural site . When environmental factors were included as predictors, the model fit for crAssphage improved (adjusted 2R = 0.62). Diurnal variability of crAssphage was significant at both sites (p < 0.001), but displayed significantly different trends (p < 0.001). In the second model, concentrations of crAssphage peaked between 17:00–21:30 at the urban site and between 04:00 and 09:30 at the natural site  at the urban site . For crAral site . For crAral site .p = 0.87) but did at the natural site . When we adjusted for the effect of environmental factors, the model fit improved (adjusted 2R = 0.45), but diurnal variability was still not detected at the urban site (p = 0.07). At the natural site, concentrations peaked between 02:00–06:30 and declined from 06:30–18:30 . Independent of environmental factors, GAMM analysis showed that blaCMY exhibited significant diurnal variability at the urban site (p = 0.043), as well as a significantly different diurnal trend at the natural site . Concentrations of blaCMY peaked between 8:00 and 12:30 at both sites (2R = 0.20). However, this model was weighted toward undetected samples and resulted in a loss of detection of diurnal variability  and a different, significant diurnal trend at the natural site . The model fit improved when we included the effect of environmental factors (adjusted 2R = 0.38). At both sites, concentrations of intI1 peaked between 6:30 and 12:30, matching the diurnal trends observed with crAssphage (For Assphage . Water tAssphage .KPC was inconsistently detected at both sites (50/93 samples were below the LOD). GAMM analysis showed that KPC exhibited significant diurnal variability at the urban site (p = 0.003), with a similar significant diurnal trend at the natural site . The temporal trend closely matched that of blaCMY, with peak concentrations of KPC occurring between mid-morning to early afternoon , indicating that environmental factors were not significantly associated with diurnal variability for KPC  are shed in the feces of a variety endo- and ectotherms and are not animal specific , or ARGs at the urban site. The lack of consistent associations between E. coli and MST markers in our study .


Cymodocea nodosa along the natural pH gradient of Levante Bay, Vulcano Island, Italy. The bacterial community was determined by characterizing the 16S rRNA amplicon sequencing and analyzing the operational taxonomic unit classification of bacterial DNA within samples. Statistical analyses were used to explore how life-long exposure to different pH/pCO2 conditions may be associated with significant differences in microbial communities, dominant bacterial classes, and microbial diversity within each plant section and sediment. The microbiome of C. nodosa significantly differed among all sample types and site-specific differences were detected within sediment and root/rhizome microbial communities, but not the leaves. These results show that C. nodosa leaves have a consistent microbial community even across a pH range of 8.15 to 6.05. The ability for C. nodosa to regulate and maintain microbial structure may indicate a semblance of resilience within these vital ecosystems under projected changes in environmental conditions such as ocean acidification.Seagrass meadows are hotspots of biodiversity with considerable economic and ecological value. The health of seagrass ecosystems is influenced in part by the makeup and stability of their microbiome, but microbiome composition can be sensitive to environmental change such as nutrient availability, elevated temperatures, and reduced pH. The objective of the present study was to characterize the bacterial community of the leaves, bulk samples of roots and rhizomes, and proximal sediment of the seagrass species The online version contains supplementary material available at 10.1007/s00248-021-01867-9. A vitalorldwide . SeagrasA functioning microbiome is vital to the physiology, ecology, and biogeochemistry of seagrass habitats , but theThe rhizosphere of seagrasses has a unique bacterial community that is typically important within the sulfur cycle and aids in shaping the overall health of the seagrass meadow , 15. The4+ into NO2− [Sediments within seagrass meadows are often an extreme environment with anoxic conditions commonly produced through the mineralization of organic material throughout the benthic layer –27. In anto NO2− , 33, mitnto NO2− , 34. Seanto NO2− –37 and anto NO2− .2 is anticipated to reach 985 ± 97 ppm (full-range 794–1142 ppm), which will concomitantly result in higher oceanic pCO2 conditions [pCO2, along with a rise in bicarbonate, allows for optimal photosynthesis [pCO2 may not be advantageous in all aspects. A study characterizing phenol production in marine plants showed a decline in concentrations of phenolic protective substance with increasing pCO2 levels [pCO2, such as near CO2 vents, and an overall negative impact on seagrasses. At extreme levels of pCO2, however, Borell et al. (2013) showed that increased pCO2 levels changed the palatability of macroalgae reducing the grazing rates of herbivory. The change in palatability was due to an increase of secondary metabolites (dimethylsulfoniopropionate) [pCO2 are also associated with a disruption in the microbiome of marine organisms. For example, seagrass within naturally high pCO2 conditions near Papua New Guinea harbored more potentially pathogenic bacteria compared with those within lower pCO2 sites [Caulerpa, an invasive macroalgae, showed a stable diversity and species richness within its microbiome under high pCO2 conditions suggesting that this species may be resistent or even potentially benefit from changing ocean conditions [Caulerpa’s chloroplasts aids in intracellular pCO2 regulation under high pCO2 conditions, giving them a competitive advantage over macroalgae that lack a CCM [pCO2 and, concomitantly, low pH on seagrasses and their microbial communities still require further investigation as elevated pCO2 levels potentially pose a threat to seagrass ecosystems.Global oceans are changing. By 2100, the average atmospheric COnditions . High pCynthesis . Althoug2 levels . Phenol 2 levels –50. Thuspionate) . High leO2 sites . On the nditions . The preck a CCM . The knoThe objective of the present study was to characterize the bacterial communities associated with seagrass leaves, bulk samples of roots and rhizomes, and the proximal sediments found along the natural pH gradient of Levante Bay, Vulcano Island, Italy. There are few available study sites that generally mimic current and anticipated extreme conditions within one naturally occurring community. Volcanic vents contain minimal confounding variables and a natural pH gradient that allows for characterizing the present and potentially future bacterial communities of marine organisms such as seagrasses. Levante Bay located at Vulcano Island, Italy, provided a natural pH gradient to study microbial differences in a seagrass community from long-term exposure to different environmental conditions.Cymodocea nodosa and associated sediment samples were collected on June 21, 2013, in Levante Bay, Vulcano Island , NE of Sicily, Italy. Levante Bay contains a natural seawater pH gradient ranging from 6.05 to 8.15 due to the presence of a shallow-water submarine volcanic CO2 vent [NBS was measured at the time of sampling using a YSI 556 handheld multimeter . Our study did not measure other water quality information during the sampling period, but several other studies have characterized this study site. To date, there is no literature published on the trace elements along the gradient at Vulcano Island. However, two studies have published records on total alkalinity, pCO2, filamentous algae, suspended particulate organic matter, macroalgae, microbial mats, dry bulk density of sediment (g cm−3), porosity (φ), silt and clay (%), δ13C (‰), and C/N  leaves, and (2) roots and rhizomes, were collected at each site (one ramet per clump). Root and rhizomes have known differences in microbial composition [n = 3 per site) were stored separately in the RNA stabilization reagent RNAlater (Qiagen®) and then flash-frozen using liquid nitrogen. Enough RNA later was used to completely submerge all sample types. Although sediments were stored in separate sterile centrifuge tubes, the leaves and roots and rhizomes of the seagrasses of each ramet were not separated until sample processing occurred. Samples were transported back to Mote Marine Laboratory  via a liquid nitrogen dry shipper and stored at − 80 °C until further analysis.Three individual clumps of position , 57. Howwww.mrdnalab.com, MRDNA, Shallowater, TX, USA) with a single-step 30 cycle PCR, using primers specific for the 16S gene (27F and 519R). Samples were then sequenced using a Roche 454 FLX titanium sequencer as per the manufacturer’s guidelines.Nucleic acid extraction for all samples was carried out using the commercially available PowerSoil® DNA isolation kit (Qiagen®). Sediment samples were processed as per the manufacturer’s instructions. A modified protocol was used for seagrass tissues. Specific tissues  were separated under a sterile hood using sterile scalpels. The initial lysing step for the isolation kit required both a lysing solution and mechanical bead beating for 10 min, which was extended to 1 h in order to maximize DNA yields. The initial bead beating time of 10 min, resulted in an average DNA yield of 5.4 ng/µL and a range of 0–29 ng/µL with 5 samples < 1 ng/µL. The elongated bead beating increased the average DNA yield to 8.2 ng/µL and improved the range to 2.4–29 ng/µL. Samples were not periodically cooled on ice during the elongated vortexing period; however, the average DNA strand length of our sequences (~ 450 bp) showed formidable quality even after the extended bead beating time. Extracted DNA was stored at − 20 °C until 16S rRNA amplicon sequencing. Bacterial communities of each sample were analyzed using 454 pyrosequencing of the 16S rRNA gene. Samples were sequenced by MRDNA . MR DNA trimming was completed after an FLX sequencing run followed by a quality filtering of any reads less than 150 bp. From the amplicon reads, quality sequences were binned into OTUs  based on sequence similarity with a 97% similarity cutoff. OTUs were classified at all taxonomic levels using the curated NCBI database (www.ncbi.nlm.nih.gov) [Amplicon reads were analyzed by the MRDNA lab (nih.gov) . ClassifOnly sequence reads identified as bacteria were processed through the statistical analyses. Bacterial community data were rarefied to 3036 reads per sample prior to analysis. Rarefication was chosen based on the lowest number of reads per sample within the data set (3036 reads). Community analyses were then conducted at the operational taxonomic unit (OTU) level. A permutational multivariate analysis of variance (PERMANOVA) with 9999 permutations  was usedA Kruskal–Wallis (KW) test was used to determine whether there were differences within dominant bacterial classes  among sites within each sample type. If a significant difference was determined using the KW test, the analysis was proceeded with a Bonferroni-corrected Dunn’s test for post hoc comparison within the dunn. test package in R  to deterAlpha diversity was calculated using the Shannon Index to compare the bacterial OTU diversity among sites within each community type . A KW test with a Bonferroni-corrected Dunn’s test was used for post hoc comparison to determine significant differences in bacterial diversity between sites.Amplicon DNA sequencing for seagrass leaves , roots and rhizomes , and sediment  resulted in a total of 529,343 total sequence reads. These sequences were binned into 7759 distinct OTUs. The shortest sequence obtained was 150 bp and the longest was 670 bp with an average length of each sequence read being 435 ± 1.37 bp , with little to no overlap of sample types within the NMDS plot .There was a significant difference in bacterial communities detected among sample types  with some overlap within the NMDS ordination plot , “vent” and “site 2” , “vent” and “site 3” , and “site 2” and “site 3” . When comparing the bacterial community of sediments among sites, bacterial OTUs found within the “vent” site were so specific to that location  it prevented NMDS plot interpretation . The pairwise PERMANOVA of the bacterial community within the sediments, however, showed no significant differences among sites. Detailed pairwise PERMANOVA comparison results can be found in Supplemental Table When the bacterial communities were compared within sample types, there were no differences among sites for seagrass leaves   Fig. .Fig. 3CBetaproteobacteria was the most abundant bacterial class at each site throughout the leaf community. The bacterial community of leaves at “site 1” had two dominant bacterial classes, Betaproteobacteria (85.77% SE ± 3.67) and Gammaproteobacteria (8.87% SE ± 0.98). Alphaproteobacteria and Deltaproteobacteria were major contributors to the bacterial communities of leaves at “site 2”, “site 3,” and “vent,” but not at the “reference” site nor at “site 1.” Out of the six dominant classes within the leaf community, two classes were dominant in only one site location. Oscillatoriophycideae was specific to the Reference site and an unknown Cyanobacterium was specific to “site 2.”Six different bacterial classes dominated the leaf samples Fig. . BetaproOscillatoriophycideae  was significantly higher at the “reference” site compared to that at both “site 1” (p = 0.02) and “site 3” (p = 0.02). These significant differences were driven primarily by OTU 2  , which showed significantly higher abundances at the “reference” site compared with all other sites . Bacterial classes Bacteroidia (3.18% SE ± 0.59) and Clostridia (3.82% SE ± 1.34) were only dominant within “site 3” of the root community. Subsection II Cyanobacteria was only dominant within the root samples from the “vent” site. Oscillatoriophycideae was present within the “reference” site of the root community (21.49% SE ± 6.74).The bacterial community of the roots and rhizomes was dominated by nine different classes. Three of those classes were specific to the root samples: mes Fig. . BetaproBacteroidia , Clostridia , Oscillatoriophycideae , and an unknown Cyanobacterium . Bacteroidia showed a significantly higher abundance at “site 3” compared to the “reference” site (p = 0.02) and “vent” site (p = 0.01). Clostridia was significantly higher at “site 3” than at both the “reference” site (p = 0.02) and the “vent” site (p = 0.02). Oscillatoriophycideae was significantly higher at the “reference” site compared to “site 1” (p = 0.04), “site 2” (p = 0.01), and “site 3” (p = 0.04). Similar to leaves, OTU 2  showed significantly higher abundance at the “reference” site compared to all other sites . Bacteroidia, Clostridia, and the unknown Cyanobacterium had no significant OTUs that differed among sites, which could have explained the differences detected at the class level. However, several other OTUs significantly differed among sites , where Alphaproteobacteria was the most abundant (21.71% SE ± 1.55). Bacterial classes that were only detected at specific sites included Acidimicrobiia at the “reference” site (3.50% SE ± 0.37), Cytophagia at both “reference” (3.10% SE ± 0.13) and “site 2” (5.36% SE ± 3.90), Candidatus Thiobios at the “vent” site (6.92% SE ± 0.77), and both Sphingobacteria (3.75% SE ± 0.36) and Zetaproteobacteria at “site 2” (5.78% SE ± 0.64). Planctomycetacia was present in all sites except for the “vent” site and Deltaproteobacteria was present in all sites.The bacterial community of the sediments was dominated by eleven different bacterial classes Fig. . ClassesAcidimicrobiia  was significantly higher at the “reference” site when compared to that at the “vent” site (p = 0.01), primarily driven by a single OTU, Acidimicrobiia’s OTU 157  (p = 0.02). Phylum Proteobacteria’s, Candidatus Thiobios (OTU 46)  was significantly higher at the “vent” site compared with the “reference” site (p = 0.007). Gammaproteobacteria  was significantly higher in abundance at the “vent” site when compared with that in the “reference” site (p = 0.04) and “site 2” (p = 0.01). Gammaproteobacteria had 3 OTUs within its classification that differed significantly among sites: OTU 16 (g. Thiothrix) , OTU 40  , and OTU 1368  . OTU 16 was significantly higher at the “vent” site compared to that at all other locations (p = 0.02). OTU 40 was only significantly higher at the “vent” site compared to that at “site 2” (p = 0.02). OTU 1368 was also significantly higher at the “vent” site compared with that at “site 2”  was significantly different among sites, but the post hoc analysis failed to determine any significant differences among the sites. Sphingobacteria  was significantly higher at “site 2” when compared with that at the “vent” site (p = 0.01). Zetaproteobacteria  was significantly higher at “site 3” compared to that at the “vent” site (p = 0.01). Planctomycetacia, Sphingobacteria, and Zetaproteobacteria had no significant OTUs that differed among sites. The average relative abundance of each bacterial OTU within the sediment community that differed significantly among sites can be found in Supplemental Fig. Seven of the eleven dominant classes showed significant differences among sites. C. nodosa leaves, roots rhizomes, and associated sediments were significantly different from each other. The bacterial community within the rhizosphere is influenced by plant metabolites [In the present study, bacterial communities of abolites  and may abolites , suggestpCO2. However, our study did not incorporate water samples for microbiome analysis so this apparent stability may be a reflection of local bacterial communities within the aqueous environment. These results, however, do suggest that below-ground ecology might be influenced more by environmental change compared to above-ground ecology. Ettinger et al. (2017) showed a similar site-to-site difference in the sediment bacterial community, but not within the seagrass roots and rhizomes. Their study, however, characterized the microbial community in proximity to the seagrass patch and did not focus on changing environmental factors [pCO2 levels for C. nodosa at volcanic seeps in Greece and Italy, suggesting a greater impact of environmental factors on the below-ground ecology for this species of seagrass. The altered below-ground ecology in Mishra (2018) was also associated with a decline in C. nodosa survivability with increasing pCO2 due to reduced nutrient availability and soil toxicity [pCO2, appear to have the greatest influence on the bacterial community of sediments, followed by the roots, and eventually the leaves if conditions become extreme [pCO2. Duarte et al. (2018) showed marine macrophytes contain the ability to acclimate their hologenome under typically stressful ocean conditions [C. nodosa leaves as the bacterial communities associated with the C. nodosa leaves were most similar among sites. Currently, seagrass species that can regulate their microbiome under different environmental conditions appear to be limited to the leaves and, to a lesser extent, roots and rhizomes. Further investigation into the algal exudates potentially responsible for selecting bacteria should be considered to determine the degree of influence on the microbial community of the proximal sediment.There were site-specific differences in bacterial communities of roots and rhizomes and sediments, but not in the leaf community, which remained consistent along the gradient. These results suggest that leaves may provide a more stable environment than roots, rhizomes, and sediment under changing levels of  factors . The addtoxicity . However extreme , 64. Sea extreme , which dnditions . Indeed,nditions , 67. In Betaproteobacteria (37.88%), Gammaproteobacteria (18.59%), and Alphaproteobacteria (14.05%) were the most abundant classes of bacteria among all samples at Levante Bay. Another study, which also quantified the bacterial communities of sediments within Levante Bay, showed the same dominant classes of bacteria to be the most abundant [Firmicutes, Bacteroidetes, and Actinobacteria to be dominant as well. Alternatively, the present study detected several other taxa that were relatively common within the sediments that were not found in Kerfahi et al. (2014) including Zetaproteobacteria, Sphingobacteria, Planctomycetecia, Flavobacteria, Cytophagia, Candidatus Thiobios, and Acidimicrobia. Differences in the bacterial classes detected between these two studies could be ecologically or methodologically driven. Further longitudinal studies that utilize the same methods through time would better characterize the potential temporal changes of the sediment microbial structure within Levante Bay. Additionally, changes occur at different taxonomic scales. Analyzing bacteria at multiple taxonomic resolutions through time may elucidate further influences of environmental conditions on sediment microbiota.The present study showed that overall abundant . HoweverBetaproteobacteria regardless of the site, but this bacteria taxon was significantly lower within site 2 (pH 7.86) compared with the other sites. Similarly, a drop in Betaproteobacteria at site 2 was also recorded in the sediment. Betaproteobacteria plays an essential role in stabilizing the microbial community through the regulation of harmful algae [Betaproteobacteria also is an ammonia-oxidizing bacteria (AOB) that plays an important role in nitrification. AOB abundance and nitrification rates have shown seasonal variability due to changing salinities [C. nodosa. The unique microbial community of the leaves and sediment sample types within site 2 suggests a concomitantly unique environmental condition within this location that warrants further exploration. Additionally, there was a decline in Betaproteobacteria at the vent site for all sample types, further indicating that this bacterium, a part of the C. nodosa core microbiome, may be susceptible to extreme pCO2 conditions.The bacterial community of the leaves was heavily dominated by ul algae . Betaprolinities . HoweverOscillatoriophycideae — phylum Cyanobacteria — abundance within the “reference” site compared with the other sites with lower pH conditions. In addition, the biofilm on seagrasses within another naturally occurring CO2 vent in Papua New Guinea showed a significant reduction in the relative abundances and diversity of cyanobacteria closer to the vent compared with control sites [2 vents. Nitrogen-fixing cyanobacteria are prevalent on seagrass leaves. However, they are also found to inhabit other sections of the plant, which includes the rhizomes that do not receive light [Oscillatoriales . Differences among classes in sediment samples were driven by just five OTUs, o. Acidimicrobiales, o. Candidatus Thiobios, g. Thiothrix, sp. Balneatrix balneatrix, and g. Balneatrix. This suggests that a more detailed analysis at the OTU level could either reveal additional meaningful nuances within the data or lead to large levels of variation that mask trends at greater taxonomic levels. For instance, two OTUs belonging to o. Candidatus Thiobios and g. Thiothrix were significantly higher at the vent site. The higher abundance of these two OTUs is likely due to their sulfur oxidative properties, which are important for carbon fixation in pCO rich environments and are common bacteria surrounding hydrothermal vents [Balneatrix at vent sites may be due to groundwater discharge from the Vulcano Porto Aquifer [The present study documented site-specific responses at both the bacterial class and OTU level, suggesting that small-scale environmental variations not captured within the study could be influencing the bacterial microbiome. There were several OTUs that significantly differed among sites  demonstrated a shift in the sediment bacterial community under low pH conditions at Levante Bay, Italy [Gammaproteobacteria remained stable in abundance, despite the shift in more rare taxa within the bacterial community with reduced pH. Site location must be considered, nevertheless, as there were differences in both site location and pH levels within the study sites. Despite these differences, our study and that of Kerfahi (2014) show stability within the sediment microbial community for dominant bacteria such as Gammaproteobacteria before extreme conditions are reached. Both studies also highlight the potential for disruption within the community of rare taxa [. Alternatively, Krause et al. (2012) suggest sediment microbial communities were less stable in the North Sea with moderate changes in pH resulting in significant alterations to the microbial assemblage [The species richness of the microbial community was not significantly different among sample types or sites, but previous studies showed that epibiont communities increase in species richness with declining pH levels creating a more complex microbial community . Previouy, Italy . Howeverare taxa . Alternasemblage . Despite Kerfahi 014 demonsemblage .C. nodosa may regulate its microbial community in the face of extreme pH conditions. The results also suggest that the surrounding environment may disrupt the microbiome of the seagrass root structures as well as the sediments themselves more so than the leaves. However, to date, there is no complementary study that assesses the bacterial turnover rates for C. nodosa with associated water samples through time. A study such as this could further elucidate the spatial and temporal variations of the microbiome within seagrass habitats. Characterizing the potential shifts in the microbial community of seagrasses under varying environmental conditions will be vital to determine the physiological ramifications of global change on this foundational taxonomic group.Characterizing the different microbiome structures under natural environmental gradients, such as that in Levante Bay, is critical to providing insight into how a major component of organismal physiology, the host microbiome, will change under future ocean conditions. The results of the present study suggest Supplementary file1 (DOCX 2567 KB)Below is the link to the electronic supplementary material.


A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC. In eukaryotic cells, mitochondria are the site of oxidative metabolism reactions, including the citric acid cycle, the β-oxidation of fatty acids, and oxidative phosphorylation. Although the mitochondrial outer membrane is permeable to small molecules due to the presence of transport proteins called porins , the innThe biosynthesis of heme is an example of a multistep process which requires tight regulation of mitochondria and cytosol to assure the presence of heme precursors and heme itself, crucial both for heme prosthetic group biosynthesis and for the assembly of heme proteins ,11,12,13Some studies have proposed that the accumulation of porphyrin derivatives could be mediated by a carrier-transport-like mechanism since porphyrin derivatives are anions, slightly negatively charged at physiological pH, making it difficult for them to cross the inner mitochondrial membrane by passive diffusion ,15,16.In heme biosynthesis, the ATP-binding cassette (ABC) transporter sub-family B member 6 (ABCB6) and the mitochondrial peripheral-type benzodiazepine receptor (PBR) have been identified as the best candidates for transporting porphyrins across the outer membrane ,17,18. Interestingly, many metabolite transporters of the inner mitochondrial membrane, known as mitochondrial carriers SLC25A family) 5A family,7,8,9, hAmong mitochondrial carriers, the OGC is one of the most studied carriers and plays an important role in the malate/aspartate shuttle ,24,25. ICxRK, which is shared by most of the MCs in at least one repeat. This motif is located at the level of an MC’s matrix loops and the adjacent short-helices parallel to the membrane plane, between the two portions of the conserved MC signature motif sequence PXD/EXXK/R…..EGXXXXArK/RG  2-oxoglutarate and [14C] malate were purchased from Perkin-Elmer Life Sciences . All other chemicals used were of analytical grade. Hydroxylapatite (Bio-gel HTP) and Amberlite Bio-Beads SM-2 was obtained from Bio-Rad; Matrex Gel Orange was obtained from Amicon ; Triton X-100, Triton X-114, acrylamide, and N,N′-methylenebisacrylamide were obtained from Serva; egg-yolk phospholipids were obtained from Fluka; hemin, cardiolipin, 1,4-piperazine-diethanesulphonic acid (Pipes), Sodium dodecyl sulfate, (SDS) and asolectin were obtained from Sigma; celite 535 was obtained from Roth; Sephadex G-75 was obtained from Pharmacia; 2-oxoglutarate or [14C] malate uptake  = −KEThe double reciprocal graph of 14C] malate uptake was investigated as a function of different external hemin concentrations in exchange with the internal 2-oxoglutarate in proteoliposomes reconstituted with the purified OGC. This was in line with the main physiological role of the OGC carrier in the brain, where it is maximally efficient in catalyzing the exchange between the external malate and internal 2-oxoglutarate  malate were used, and the results of Thus, different concentrations of  malate influx in proteoliposomes containing hemin was evaluated as a function of the malate concentrations, the double reciprocal plot showed straight lines which met in the third quadrant of the diagram  of external substrate “A” [emin “E” .14C] malate) and hemin characterizing the fast stages of the transport process , and not the 2-oxoglutarate (B), was monitored.Notably, as shown below, the equilibrium constant relating to the ternary ECB complex was not reported because the uptake the e can be expressed as functions of the concentration of the substrate, the equilibrium constants, and the kinetic constants, deriving the following equation:T, were equal to the contribution of all species involved in the transport process:Overall, in the steady state in which the transport rate remained constant, the concentrations of the different forms of the carrier also remained stationary, depending on the various listed constants, as well as on the concentration of the substrate and hemin. Therefore, using the above relations, [CA]i and Gi were also introduced as functions of the concentration of B within the proteoliposomes and were therefore constant as long as the concentration of B was kept constant:For further simplification, FThus, it was necessary to verify that the experimental results fit the general theoretical equation and those derived from them to demonstrate the hypothesis that hemin was a partially competitive inhibitor of the transport of the malate in the OGC carrier reconstituted in proteoliposomes. It was also necessary to try to understand whether hemin binds the carrier from the same face of the entering radiolabeled substrate (as described in ), from tStarting from the general Equation (1), the transport rate, V, can be expressed (1) as a function of [A], the external concentration of the malate, maintaining [E], the external concentration of hemin, as a constant, and (2) the transport rate, V, can be expressed as a function of [E] when [A] is kept constant. M and KM were respectively equal to:In the first case, for each concentration of E, the equation describing the Michaelis-Menten trend was obtained and, therefore, the graph of double reciprocal met on the ordinate axis. In other words, we obtained hyperbola whose equation was:To simplify the expression, the equilibrium constants M was independent from [E], while the KM was not  was expressed as a function of the concentration of E, where the concentration of A was kept constant, the equation of rate V was expressed as a function of [E], which described a decreasing hyperbolic graph represented by the equation below.0.5, varied in function of the concentration of A. Additionally, V0, varied in function of the concentration of A and was defined by the expression:VV0 and K0.5 were always function of the external concentration of malate (A), the asymptote represented the abscissa axis at any concentration of the malate.In this case, while the parameters V14C] malate in proteoliposomes was measured as a function of increasing concentration of hemin in the presence of three different labeled malate concentrations (0.025–0.1 mM), our experimental data fit Equation (3) rather than (5). In fact, three decreasing hyperbolic curves were obtained  ,74. Ther