Phenotypic Methods Research Articles

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Prevalence of blaOXA-10, blaCTX-M-3 and SHV Genes among ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical samples in Basra city, Iraq

The current study was conducted to determine the prevalence of extended-spectrum β-lactamase (ESBL) in 78 drug-resistant clinical isolates (25 Klebsiella pneumoniae and 53 Escherichia coli strains) using phenotypic and molecular methods. The phenotypic method was performed using a double-disk synergy test (DDST), while the genotypic method screened for the blaSHV, blaCTX-M13U, and blaOXA-10 genes using specific primers. The phenotypic results showed that out of 53 tested strains of E. coli, 17 (32.07%) produced ESBL. Similarly, out of 25 tested strains of K. pneumoniae, 8 (32%) produced ESBL. Genotypic detection showed that in E. coli, the most abundant gene was SHV, present in 24 strains (45.28%), followed by blaOXA-10 in 23 strains (43.39%) and CTX-M-3 in 8 strains (15.09%). In K. pneumoniae, SHV was detected in 12 strains (48%), followed by OXA-10 and CTX-M-3, each found in 5 strains (20%).

A comparative evaluation of five phenotypic methods for identification of carbapenemase-producing Enterobacteriaceae: a modified carbapenemase detection test.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) is urgently needed to prevent their spread in healthcare settings. Here, we have evaluated the performance of the phenotypic methods for detection of carbapenemase production directly from bacterial cultures. A total of 99 clinical and rectal Enterobacteriaceae isolates were included (81 carrying known carbapenemase-encoding genes and 18 without carbapenemase production). All isolates were subjected to the five phenotypic tests including in-house Carba NP (iCarba NP), modified-Carba NP, E-Test MBL, modified Hodge test (MHT), and commercial combination disk test. Test results were read at different time points for iCarba NP and modified-Carba (1 min, 5 min, 15 min, 1 h and 2 h). The sensitivity and specificity of the iCarba NP were 78.87% and 100%, respectively, whereas those of the modified-Carba NP test were 95.06% and 94.44%, respectively. False-negative results were detected in four OXA-48 isolates with the use of modified-Carba NP, whereas one non-carbapenemase isolate had false-positive results. The sensitivity/specificity was 91.30%/100% and 80.25%/83.33% for the E-Test MBL and MHT, respectively. The sensitivity and specificity of the aminophenylboronic acid synergy test were 100% and 97.94%, respectively, whereas those of the dipicolinic acid synergy test were 82.61% and 96.23%, respectively. Rapid, simple, and reliable methods are needed for laboratory detection of CPE isolates to improve the detection and surveillance of these clinically relevant pathogens in an epidemiological context. We conclude that the modified-Carba NP test can be one of the reliable tests for the prediction of carbapenemase-producing bacteria.IMPORTANCEThe emergence of carbapenem resistance among Gram-negative bacteria is a serious global health threat. Here, we investigate the performance of the five phenotypic assays against carbapenemase-producing and carbapenemase-non-producing Enterobacteriaceae. Accurate and rapid detection of CPE isolates is critically required for clinical management and treatment of infections caused by these organisms. Among the five evaluated phenotypic tests, the mCNP test presented the highest sensitivity (95.06%) and, therefore, can be considered the best test to be used as a screening phenotypic methodology.

In vitro assessment of newer colistin-sparing antimicrobial agents for clinical isolates of carbapenem-resistant organisms

ObjectivesCarbapenem-resistant organisms (CROs) are a significant public health threat globally, particularly in countries like India with high antibiotic resistance rates. The current study investigates the prevalence of CROs, detects resistance mechanisms using phenotypic methods and assesses the efficacy of newer antibiotics against CROs. MethodsA prospective study conducted at a tertiary care hospital in India during 2021–23. Clinical specimens were processed and bacterial identification was performed using MALDI-TOF mass spectrometry followed by antimicrobial susceptibility testing using CLSI guidelines against a plethora of newer antibiotics for CROs. Carbapenemase production was detected using phenotypic methods, and the presence of the mcr-1 gene was assessed by real-time PCR. ResultsDuring the study period, predominantly (70 %) Gram-negative bacteria were isolated; amongst which 5692 strains were carbapenem-resistant, wherein resistance to different carbapenems ranged from 34.1% to 79 %. Majority of the carbapenemase producers were metallo-β-lactamases (MBL) producers (75 %). Colistin resistance was noted in 5.4 % of selected carbapenem-resistant isolates. Among newer antibiotics, cefiderocol demonstrated the lowest resistance rates (0–14 %), while resistance to newer β-lactam/β-lactamase inhibitor combinations was very high in carbapenem-resistant isolates. Fosfomycin, minocycline and tigecycline, each showing variable efficacy depending on the site of infection. Moreover, newer β-lactam/β-lactamase inhibitor combinations offer restricted benefits due to widespread prevalence of MBL in the region. ConclusionThe escalating prevalence of CROs in India underscores the urgency for alternative treatment options beyond colistin. Hence, highlighting the critical importance of developing effective strategies to combat carbapenem resistance.

Multi-scale characterisation of cold response reveals immediate and long-term impacts on cell physiology up to seed composition in sunflower.

Early sowing can help summer crops escape drought and can mitigate the impacts of climate change on them. However, it exposes them to cold stress during initial developmental stages, which has both immediate and long-term effects on development and physiology. To understand how early night-chilling stress impacts plant development and yield, we studied the reference sunflower line XRQ under controlled, semi-controlled and field conditions. We performed high-throughput imaging of the whole plant parts and obtained physiological and transcriptomic data from leaves, hypocotyls and roots. We observed morphological reductions in early stages under field and controlled conditions, with a decrease in root development, an increase in reactive oxygen species content in leaves and changes in lipid composition in hypocotyls. A long-term increase in leaf chlorophyll suggests a stress memory mechanism that was supported by transcriptomic induction of histone coding genes. We highlighted DEGs related to cold acclimation such as chaperone, heat shock and late embryogenesis abundant proteins. We identified genes in hypocotyls involved in lipid, cutin, suberin and phenylalanine ammonia lyase biosynthesis and ROS scavenging. This comprehensive study describes new phenotyping methods and candidate genes to understand phenotypic plasticity better in response to chilling and study stress memory in sunflower.

Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging.

Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR.

The association of TNF inhibitor use with incident cardiovascular events in radiographic axial spondyloarthritis

BackgroundWhether tumor necrosis factor inhibitor (TNFi) use is cardioprotective among individuals with radiographic axial spondyloarthritis (r-axSpA), who have heightened cardiovascular (CV) risk, is unclear. We tested the association of TNFi use with incident CV outcomes in r-axSpA. MethodsWe identified a r-axSpA cohort within a Veterans Affairs database between 2002 and 2019 using novel phenotyping methods and secondarily using ICD codes. TNFi use was assessed as a time-varying exposure using pharmacy dispense records. The primary outcome was incident CV disease identified using ICD codes for coronary artery disease, myocardial infarction or stroke. We fit Cox models with inverse probability weights to estimate the risk of each outcome with TNFi use versus non-use. Analyses were performed in the overall cohort, and separately in two periods (2002–2010, 2011–2019) to account for secular trends. ResultsUsing phenotyping we identified 26,928 individuals with an r-axSpA diagnosis (mean age 63.4 years, 94 % male); at baseline 3633 were TNFi users and 23,295 were non-users. During follow-up of a mean 3.3 ± 4.2 years, 674 (18.6 %) TNFi users had incident CVD versus 11,838 (50.8 %) non-users. In adjusted analyses, TNFi use versus non-use was associated with lower risk of incident CVD (HR 0.34, 95 % CI 0.29–0.40) in the cohort overall, and in the two time periods separately. ConclusionIn this r-axSpA cohort identified using phenotyping methods, TNFi use versus non-use had a lower risk of incident CVD. These findings provide reassurance regarding the CV safety of TNFi agents for r-axSpA treatment. Replication of these results in other cohorts is needed.

Phenotypic and molecular characterization of extended spectrum- and metallo- beta lactamase producing Pseudomonas aeruginosa clinical isolates from Egypt.

Antimicrobial resistance among Pseudomonas aeruginosa (P. aeruginosa), a leading cause of nosocomial infections worldwide, is escalating. This study investigated the prevalence of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) among 104 P. aeruginosa clinical isolates from Alexandria Main University Hospital, Alexandria, Egypt. Antimicrobial susceptibility testing was performed using agar dilution technique, or broth microdilution method in case of colistin. ESBL and MBL prevalence was assessed phenotypically and genotypically using polymerase chain reaction (PCR). The role of plasmids in mediating resistance to extended-spectrum β-lactams was studied via transformation technique using plasmids isolated fromceftazidime-resistant isolates. Antimicrobial susceptibility testing revealed alarming resistance rates to carbapenems, cephalosporins, and fluoroquinolones. Using PCR as the gold standard, phenotypic methods underestimated ESBL production while overestimating MBL production. Eighty-five isolates (81.7%) possessed only ESBL encoding genes, among which 69 isolates harbored a single ESBL gene [blaOXA-10 (n = 67) and blaPER (n = 2)]. Four ESBL-genotype combinations were detected: blaPER + blaOXA-10 (n = 8), blaVEB-1 + blaOXA-10 (n = 6), blaPSE + blaOXA-10 (n = 1), and blaPER + blaVEB-1 + blaOXA-10 (n = 1). Three isolates (2.9%) possessed only the MBL encoding gene blaVIM. Three ESBL + MBL- genotype combinations: blaOXA-10 + blaAIM, blaOXA-10 + blaVIM, and blaPER + blaOXA-10 + blaAIM were detected in 2, 1 and 1 isolate(s), respectively. Five plasmid preparations harboring blaVEB-1 and blaOXA-10 were successfully transformed into chemically competent Escherichia coli DH5α with transformation efficiencies ranging between6.8 × 10 3 and3.7 × 10 4 CFU/μg DNA plasmid. Selected tested transformants were ceftazidime-resistant and harbored plasmids carrying blaOXA-10. The study highlights the importance of the expeditious characterization of ESBLs and MBLs using genotypic methods among P. aeruginosa clinical isolates to hinder the development and dissemination of multidrug resistant strains.

Coagulase-negative staphylococci enhance the colour of fermented meat through a complex cross-talk between the arginase and nitric oxide synthase activities

The nitric oxide synthase (NOS) activity was investigated in 35 staphylococcal strains from five distinct coagulase-negative staphylococci (CNS) species. NOS pathway is related with the production of nitric oxide, potentially leading to the formation of nitrosomyoglobin imparting a pinkish-red colour to fermented meats. NOS activity was investigated employing both genotypic and phenotypic methods. Among the strains studied, 16 were found to contain the gene encoding NOS, while 25 strains possessed the arginase gene. Strains from Staphylococcus saprophyticus, S. equorum, and S. carnosus species exhibited phenotypic NOS-like activity. This was evidenced by the production of l-citrulline and a comparably lower release of l-ornithine in meat simulation media fermented under microaerobic conditions by those staphylococcal cultures. The authentic NOS activity was further examined using lab-scale meat models. The characteristic red colouration, typically associated with sodium nitrite-cured meat products, was observed. Strains L31, L49, and V10 (S. saprophyticus) demonstrated a pronounced effect on meat colouration, achieving shades comparable to nitrite-enhanced meat; however, a concurring up-accumulation of putrescine and spermine (likely originating from ornithine) was also outlined. These findings suggest the potential of meat-associated CNS to promote colour development in nitrite-free meats, offering a promising area for enhancing the visual appeal of such products.

Characterization of carbapenemase-producing Enterobacterales from rectal swabs of patients in the intensive care units of a tertiary hospital in Cali-Colombia

BackgroundCarbapenemase-producing Enterobacterales (CPE) represents a significant threat to global health. This study aimed to characterize clinically and molecularly the CPE isolated from rectal swabs of patients in the intensive care units (ICUs) of a tertiary hospital in Cali, Colombia. MethodsThis was a cross-sectional observational study. Rectal swabs from patients admitted to the ICUs were collected. Bacterial identification and carbapenemase production were determined using phenotypic and molecular methods. Demographic and clinical data were extracted from electronic medical records. ResultsThe study included 223 patients. Thirty-six patients (36/223, 16.14 %) were found to be colonized or infected by CPE. Factors such as prolonged stay in the ICU, previous exposure to carbapenem antibiotics, use of invasive procedures, and admission due to trauma were associated with CPE. Klebsiella pneumoniae (52.5 %) was the most prevalent microorganism, and the dominant carbapenemases identified were KPC (57.8 %) and NDM (37.8 %). ConclusionDistinguishing carbapenemase subtypes can provide crucial insights for controlling dissemination in ICUs in Cali, Colombia.

Advancing selective breeding in leopard coral grouper (P. leopardus) through development of a high-throughput image-based growth trait

Utilizing image-based computer vision techniques, many high-throughput phenotyping methods have been employed to capture intricate growth trait characteristics, offering reliable estimates of phenotypic traits crucial for breeding programs. In this study, we explored the application of partial differential equation (PDE)-based level set approaches to introduce image-based body area percentage (IBAP) as a novel growth trait in Plectropomus leopardus, as a substitution for the traditional growth trait body weight (BW). Assessing the genetic parameters essential for robust growth trait improvement in P. leopardus breeding programs, we estimated SNP-based heritability for IBAP and BW using a comprehensive set of SNPs (673,039 SNPs with MAF >2%). Results revealed heritability estimates of 0.515 (S.E. 0.06) for IBAP and 0.542 (S.E. 0.06) for BW. Moreover, strong phenotypic and genetic correlations of 0.812 (S.E. 0.001) and 0.903 (S.E 0.021) between IBAP and BW, respectively, underscored the potential for IBAP as a surrogate trait of BW for the genetic improvement of P. leopardus. We established a linear regression model of IBAP and BW (y ​= ​−730 ​+ ​1700×, (R2 ​= ​0.71)), after rigorous assessments of linearity, normality, and homoscedasticity, to confirm model fit. Evaluation of breeding value prediction accuracies using two linear models (rr-GBLUP and Bayes B) and a non-linear (RKHS) model demonstrated the superior performance of RKHS across IBAP and BW. Exploring the impact of varied marker densities for SNP selection on genomic prediction accuracy for IBAP and BW demonstrated a threshold of 10,000 SNPs for maximal model accuracy. These findings provide essential reference information and methodological groundwork for leveraging image-based traits in P. leopardus breeding endeavors, facilitating more efficient and precise genetic improvement programs.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

Digital phenotyping data and anomaly detection methods to assess changes in mood and anxiety symptoms across a transdiagnostic clinical sample.

Clinical assessment of mood and anxiety change often relies on clinical assessment or self-reported scales. Using smartphone digital phenotyping data and resulting markers of behavior (e.g., sleep) to augment clinical symptom scores offers a scalable and potentially more valid method to understand changes in patients' state. This paper explores the potential of using a combination of active and passive sensors in the context of smartphone-based digital phenotyping to assess mood and anxiety changes in two distinct cohorts of patients to assess the preliminary reliability and validity of this digital phenotyping method. Participants from two different cohorts, each n = 76, one with diagnoses of depression/anxiety and the other schizophrenia, utilized mindLAMP to collect active data (e.g., surveys on mood/anxiety), along with passive data consisting of smartphone digital phenotyping data (geolocation, accelerometer, and screen state) for at least 1 month. Using anomaly detection algorithms, we assessed if statistical anomalies in the combination of active and passive data could predict changes in mood/anxiety scores as measured via smartphone surveys. The anomaly detection model was reliably able to predict symptom change of 4 points or greater for depression as measured by the PHQ-9 and anxiety as measured for the GAD-8 for both patient populations, with an area under the ROC curve of 0.65 and 0.80 for each respectively. For both PHQ-9 and GAD-7, these AUCs were maintained when predicting significant symptom change at least 7 days in advance. Active data alone predicted around 52% and 75% of the symptom variability for the depression/anxiety and schizophrenia populations respectively. These results indicate the feasibility of anomaly detection for predicting symptom change in transdiagnostic cohorts. These results across different patient groups, different countries, and different sites (India and the US) suggest anomaly detection of smartphone digital phenotyping data may offer a reliable and valid approach to predicting symptom change. Future work should emphasize prospective application of these statistical methods.

Phenotypic and genotypic characterization of methicillin and vancomycin-resistant Staphylococcus aureus isolated from human clinical samples in Kerman hospitals, Iran

Today, methicillin-resistant S. aureus (MRSA) isolates are widely spread in both communities and hospitals, causing high morbidity and mortality worldwide. However, the emergence of vancomycin resistance among MRSA isolates has been perceived as a formidable threat to therapeutic management. Therefore, the present study aimed to determine the frequency and antibiotic susceptibility of MRSA and vancomycin-resistant S. aureus (VRSA) strains in Kerman hospitals in Iran. A total of 455 human clinical samples were collected from Kerman hospitals. The samples were examined for the presence of S. aureus and the isolates were tested for their susceptibility to antibiotics by disk diffusion method. MRSA and VRSA isolates were detected by a combination of phenotypic and genotypic methods. Sixty-five coagulase-positive S. aureus strains were isolated, and the higher isolation rate was from wound samples (47.7%) compared with blood (18.5%), respiratory tract secretions (10.8%), urine (10.8%), body fluids (9.2%), abscess (1.5%), and cerebrospinal fluids (1.5%). The isolates exhibited high resistance toward tetracycline (69.2%). Thirty-four isolates (52.3%) were categorized as MRSA (phenotypically resistant to cefoxitin). mecA gene was amplified in 97.1% of MRSA strains. Moreover, only one MRSA strain was resistant to vancomycin (MIC=128), and this isolate carried the vanA gene. The frequency of MRSA was not significantly associated with gender, age, sample type, and sampling locality (p>0.05). Given the alarming rate of resistance among MRSA isolates, monitoring of antibiotic resistance should be performed to reduce treatment failure in patients with staphylococcal infections. Although vancomycin remains a drug of choice for MRSA, our study suggests that its efficacy may be limited by resistance development.

Low antiviral resistance in Influenza A and B viruses isolated in Mexico from 2010 to 2023

The most widely used class of antivirals available for Influenza treatment are the neuraminidase inhibitors (NAI) Oseltamivir and Zanamivir. However, amino acid (AA) substitutions in the neuraminidase may cause reduced inhibition or high antiviral resistance. In Mexico, the current state of knowledge about NAI susceptibility is scarce, in this study we report the results of 14 years of Influenza surveillance by phenotypic and genotypic methods. A total of 255 isolates were assessed with the NAI assay, including Influenza A(H1N1)pdm09, A(H3N2) and Influenza B (IBV). Furthermore, 827 sequences contained in the GISAID platform were analyzed in search of relevant mutations.Overall, five isolates showed highly reduced inhibition or reduced inhibition to Oseltamivir, and two showed reduced inhibition to Zanamivir in the NAI assays. Additionally, five A(H1N1)pdm09 sequences from the GISAID possessed AA substitutions associated to reduced inhibition to Oseltamivir and none to Zanamivir. Oseltamivir resistant A(H1N1)pdm09 harbored the H275Y mutation. No genetic mutations were identified in Influenza A(H3N2) and IBV. Overall, these results show that in Mexico the rate of NAI resistance is low (0.6%), but it is essential to continue the Influenza surveillance in order to understand the drug susceptibility of circulating strains.

Shape-Versatile Fixed Cellular Materials for Multiple Target Immunomodulation.

Therapeutic cells are usually administered as living agents, despite the risks of undesired cell migration and acquisition of unpredictable phenotypes. Additionally, most cell-based therapies rely on the administration of single cells, often associated with rapid in vivo clearance. 3D cellular materials may be useful to prolong the effect of cellular therapies and offer the possibility of creating structural volumetric constructs. Here, the manufacturing of shape-versatile fixed cell-based materials with immunomodulatory properties is reported. Living cell aggregates with different shapes (spheres and centimeter-long fibers) are fixed using a method compatible with maintenance of structural integrity, robustness, and flexibility of 3D constructs.The biological properties of living cells can be modulated before fixation, renderingan in vitroanti-inflammatory effect toward human macrophages, in line with a decreased activation of the nuclear factor kappa B (NF-κB) pathway that preponderantly correlated with the surface area of the materials. These findings are further corroborated in vivo in mouse skin wounds. Contact with fixed materials also reduces the proliferation ofactivated primary T lymphocytes, while promotingregulatory populations. The fixation of cellular constructs is proposed as a versatile phenotypic stabilization method that can be easily implemented to prepare immunomodulatory materials with therapeutic potential.

Identification of Konosirus punctatus (Temminic and Schlegel, 1846) stocks in Korean waters via otolith shape analysis and comparison of growth parameters

Accurate identification of fish stocks is essential for the successful management of fishery resources. In this study, the populations of dotted gizzard shad, Konosirus punctatus, which is listed on the International Union for Conservation of Nature red list of the threatened species, were identified by combining two phenotypic methods: otolith shape analysis and comparison of growth parameters. Otolith shape analysis revealed two types of otoliths, pointing to the presence of two gizzard shad stocks in two areas along the Korean coast: the East Sea and the Yellow Sea + western Korean Strait. It is also supported by the result that the two stocks are significantly different in the shape indices (SI), and asymptotic fork length (FL∞). These results may be due to the influence of several environmental factors, such as temperature, salinity, and food availability. Overall, our findings suggest that there are two phenotypic stocks of K. punctatus in the Korean coastal waters and they should be managed differently.

Multitemporal Field-Based Maize Plant Height Information Extraction and Verification Using Solid-State LiDAR

Plant height is regarded as a key indicator that is crucial for assessing the crop growth status and predicting yield. In this study, an advanced method based on solid-state LiDAR technology is proposed, which is specifically designed to accurately capture the phenotypic characteristics of plant height during the maize growth cycle. By segmenting the scanned point cloud of maize, detailed point cloud data of a single maize plant were successfully extracted, from which stem information was accurately measured to obtain accurate plant height information. In this study, we will concentrate on the analysis of individual maize plants. Leveraging the advantages of solid-state LiDAR technology in precisely capturing phenotypic information, the data processing approach for individual maize plants, as compared to an entire maize community, will better restore the maize’s original growth patterns. This will enable the acquisition of more accurate maize plant height information and more clearly demonstrate the potential of solid-state LiDAR in capturing detailed phenotypic information. To enhance the universality of the research findings, this study meticulously selected key growth stages of maize for data validation and comparison, encompassing the tasseling, silking, and maturity phases. At these crucial stages, 20 maize plants at the tasseling stage, 40 at the flowering stage, and 40 at the maturity stage were randomly selected, totaling 100 samples for analysis. Each sample not only included actual measurement values but also included plant height information extracted using point cloud technology. The observation period was set from 20 June to 20 September 2021. This period encompasses the three key growth stages of maize described above, and each growth stage included one round of data collection, with three rounds of data collection each, each spaced about a week apart, for a total of nine data collections. To ensure the accuracy and reliability of the data, all collections were performed at noon when the natural wind speed was controlled within the range of 0 to 1.5 m/s and the weather was clear. The findings demonstrate that the root mean square error (RMSE) of the maize plant height data, procured through LiDAR technology, stands at 1.27 cm, the mean absolute percentage error (MAPE) hovers around 0.77%, and the peak R2 value attained is 0.99. These metrics collectively attest to the method’s ongoing high efficiency and precision in capturing the plant height information. In the comparative study of different stem growth stages, especially at the maturity stage, the MAPE of the plant height was reduced to 0.57%, which is a significant improvement compared to the performance at the nodulation and sprouting stage. These results effectively demonstrate that the maize phenotypic information extraction method based on solid-state LiDAR technology is not only highly accurate and effective but is also effective on individual plants, which provides a reliable reference for applying the technique to a wider range of plant populations and extending it to the whole farmland.

New uricase producing Stenotrophomonas isolate recovered from a hot well in western south Egypt: Strain identification, Statistical optimization of enzyme production and In vitro application in gout treatment

Uricase, an enzyme catalyzing the breakdown of uric acid into more soluble compounds, holds significant therapeutic potential in managing hyperuricemia and associated conditions like gout. The current study intended to bioprospect new uricase producers in one of rarely studied ecological niches (ground water well that delivers water from 1500 m depth with temperature 50 °C in Al Qasr Oasis, New Valley Governorate, Egypt). Eight isolates were screened for uricase production, and the most potent was identified using phenotypic and genotypic methods as Stenotrophomonas maltophilia with 99% similarity. This is the first record about the ability of a member in the genus Stenotrophomonas to produce uricase, and was thus targeted in subsequent optimization studies. The initial activity of 7.20 U/mL was obtained in medium No. 2 after testing enzyme production in five different media. The uricase production was then enhanced using statistical design and modeling. Plackett-Burman design (PBD) was used to screen and identify the parameters influencing uricase activity. Four of the nine investigated factors were the most significant and were further optimized using Box-Behnken design (BBD). After two optimization cycles using PBD and BBD, the yield was improved to 20.60 and 25.67 U/mL, respectively, indicating a significant 3.5-fold improvement in the enzyme yield. The uricase was partially purified by ammonium sulphate 75% with 2.58-fold increase in activity (final recovered activity of 87.5%). Under polarized light microscopy, the prepared monosodium urate crystals clumps degraded at different levels of the obtained enzyme which encourages its future application in gout treatment.