High-Resolution Substrate Specificity Profiling of SARS-CoV-2 Mpro; Comparison to SARS-CoV Mpro.

The SARS-CoV-2 Mpro protease from COVID-19 cleaves the pp1a and pp2b polyproteins at 11 sites during viral maturation and is the target of Nirmatrelvir, one of the two components of the frontline treatment sold as Paxlovid. We used the YESS 2.0 platform, combining protease and substrate expression in the yeast endoplasmic reticulum with fluorescence-activated cell sorting and next-generation sequencing, to carry out the high-resolution substrate specificity profiling of SARS-CoV-2 Mpro as well as the related SARS-CoV Mpro from SARS 2003. Even at such a high level of resolution, the substrate specificity profiles of both enzymes are essentially identical. The population of cleaved substrates isolated in our sorts is so deep, the relative catalytic efficiencies of the different cleavage sites on the SARS-CoV-2 polyproteins pp1a and pp2b are qualitatively predicted. These results not only demonstrated the precise and reproducible nature of the YESS 2.0/NGS approach to protease substrate specificity profiling but also should be useful in the design of next generation SARS-CoV-2 Mpro inhibitors, and by analogy, SARS-CoV Mpro inhibitors as well.

The CRISPR/Cas system as an antimicrobial resistance strategy in aquatic ecosystems.

With the growing population, demand for food has dramatically increased, and fisheries, including aquaculture, are expected to play an essential role in sustaining demand with adequate quantities of protein and essential vitamin supplements, employment generation, and GDP growth. Unfortunately, the incidence of emerging/re-emerging AMR pathogens annually occurs because of anthropogenic activities and the frequent use of antibiotics in aquaculture. These AMR pathogens include the WHO's top 6 prioritized ESKAPE pathogens (nosocomial pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), extended-spectrum beta lactases (ESBLs) and carbapenemase-producing E. coli, which pose major challenges to the biomagnification of both nonnative and native antibiotic-resistant bacteria in capture and cultured fishes. Although implementing the rational use of antibiotics represents a promising mitigation measure, this approach is practically impossible due to the lack of awareness among farmers about the interplay between antimicrobial use and the emergence of antimicrobial resistance (AMR). Nevertheless, to eradicate these 'superbugs,' CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR associate protein) has turned out to be a novel approach owing to its ability to perform precise site-directed targeting/knockdown/reversal of specific antimicrobial resistance genes in vitro and to distinguish AMR-resistant bacteria from a plethora of commensal aquatic bacteria. Along with highlighting the importance of virulent multidrug resistance genes in bacteria, this article aims to provide a holistic picture of CRISPR/Cas9-mediated genome editing for combating antimicrobial-resistant bacteria isolated from various aquaculture and marine systems, as well as insights into different types of CRISPR/Cas systems, delivery methods, and challenges associated with developing CRISPR/Cas9 antimicrobial agents.

Pediatric Patients with Osteomyelitis and/or Septic Joint Undergoing Surgical Debridement Have Equivalent Short-Term Outcomes with or without Preoperative MRI.

The purpose of this study was to determine if short-term outcomes differed for pediatric patients with suspected musculoskeletal infection with or without a preoperative MRI. This was a multicenter, retrospective review of patients aged 0-16 years who presented with atraumatic extremity pain, underwent irrigation and debridement (I&D), and received at least one preoperative or postoperative MRI over a 10-year period. Primary outcomes were time to OR, total I&Ds, readmission rate, time from OR to discharge, and total number of MRIs. Secondary outcomes entailed the rate at which concurrent osteomyelitis was identified in patients with septic arthritis and the extent of the resulting surgical debridement. Of the 104 patients, 72.1% had a preoperative MRI. Patients with a preoperative MRI were significantly less likely to have surgery on the day of admission. No difference was found between groups regarding total I&Ds, readmission rate, time from OR to discharge, and total number of MRIs. Of the 57 patients diagnosed with septic arthritis, those with a preoperative MRI were significantly more likely to have concurrent osteomyelitis identified and to undergo bony debridement in addition to arthrotomy of the joint. In conclusion, patient outcomes are not adversely affected by obtaining a preoperative MRI despite the delay in time to OR. Although preoperative MRI can be beneficial in ruling out other pathologies and identifying the extent of concurrent osteomyelitis, the decision to obtain a preoperative MRI and timing of surgery should be left to the discretion of the treating surgeon.

Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma.

Radiation therapy is frequently used to treat cancers including soft tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in transplanted tumors, but the mechanism(s) remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, we performed bulk RNA-seq, single-cell RNA-seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and interferon-γ. CpG + RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG + RT, TCR clonality analysis suggests an increase in clonal T-cell dominance. Collectively, these findings demonstrate that RT + CpG significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft tissue sarcoma.

Factors Shifting Preference Toward Telemedicine in the Delivery of Skin Cancer Reconstruction Care.

OBJECTIVES: Identify which delivery modality for skin reconstruction care, face-to-face (FTF) in-person versus two telemedicine modalities, store-and-forward (S&F) and live video chat (LVC), is patient preferred and how cost, access, wait time, and demographics influence this preference. STUDY DESIGN: Cross-sectional survey. METHODS: A 16-question survey querying demographics and five scenario-specific preferences questions for the delivery of skin cancer reconstruction care was created and distributed via Amazon Mechanical Turk (MTurk), a crowdsourcing online marketplace, and in-person to Mohs micrographic surgery patients. RESULTS: 1394 MTurk and 55 in-person responses were included. While 82.1% of online respondents prefer FTF clinic visits, this decreases to 58.3% with an in-person visit cost (p < 0.01) and furthermore to a minority 43.5% with both an in-person visit cost and wait time (p < 0.01) despite 77.8% believing that usefulness to the surgeon would improve FTF. Both the MTurk and in-person cohorts demonstrated similar response patterns despite considerable demographic differences. Multivariable analyses revealed that telemedicine was preferred by MTurk respondents with Medicaid (adjusted OR [95% CI]: 1.97 [1.18-3.31]) or Medicare (1.69 [1.10-2.59]) versus private insurance, and prior skin cancer (2.01 [1.18-3.42]) and less preferred by those earning $140,000+ per year (0.49 [0.29-0.82]) compared to those earning <$20,000 per year. CONCLUSIONS: FTF visits are preferred for skin cancer reconstruction care; this shifts toward virtual care with a cost and wait time in spite of the perceived quality of care. Individuals with socioeconomic barriers to access prefer telemedicine. MTurk can be a valuable tool for behavioral research in FPRS. LEVEL OF EVIDENCE: NA Laryngoscope, 133:294-301, 2023.

Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freezing and phytopathogen susceptibility.

The model forage crop, Brachypodium distachyon, has a cluster of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were targeted for knockdown using a cold-induced promoter from rice (prOsMYB1R35) to drive miRNA. The transgenic lines showed no apparent pleiotropic developmental defects but had reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, and leaf infrared thermography. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8°C was reduced by half or killed entirely, depending on the line, compared with cold-acclimated wild type plants. In addition, more leaf damage was apparent at subzero temperatures in knockdowns after infection with an ice nucleating pathogen, Pseudomonas syringae. Although antifreeze proteins have been studied for almost 60 years, this is the first unequivocal demonstration of their function by knockdown in any organism, and their dual contribution to freeze protection as well as pathogen susceptibility, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced pathogen and freezing tolerance.

Long-Term Outcomes for Patients With Atypical or Malignant Meningiomas Treated With or Without Radiation Therapy: A 25-Year Retrospective Analysis of a Single-Institution Experience.

Purpose: Atypical (World Health Organization [WHO] grade 2) and malignant (WHO grade 3) meningiomas have high rates of local recurrence, and questions remain about the role of adjuvant radiation therapy (RT) for patients with WHO grade 2 disease. These patients frequently require salvage therapy, and optimal management is uncertain given limited prospective data. We report on the long-term outcomes for patients with atypical and malignant meningiomas treated with surgery and/or RT at our institution. Methods and Materials: Data were collected through a retrospective chart review for all patients with WHO grade 2 or 3 meningiomas treated with surgery and/or RT at our institution between January 1992 and March 2017. Progression-free survival (PFS) and overall survival (OS) were described using the KaplanMeier estimator. The outcomes in the subgroups were compared with a log-rank test. A Cox proportional hazards model was used for the univariable and multivariable analyses of predictors of PFS. Results: A total of 66 patients were included in this analysis. The median follow-up was 12.4 years overall and 8.6 years among surviving patients. Fifty-two patients (78.8%) had WHO grade 2 meningiomas, and 14 patients (21.2%) had WHO grade 3 disease. Thirty-six patients (54.5%) were treated with surgery alone, 28 patients (42.4%) with surgery and adjuvant RT, and 2 patients (3%) with RT alone. Median PFS and OS were 3.2 years and 8.8 years, respectively. PFS was significantly improved with adjuvant RT compared with surgery alone (hazard ratio, 0.36; 95% confidence interval, 0.18-0.70). Patients with Ki-67 index >10% showed a trend toward worse PFS compared with patients with Ki-67 ≤10% (hazard ratio, 0.51; 95% confidence interval, 0.25-1.04). No significant differences in PFS or OS were observed with respect to Simpson or WHO grade. Conclusions: For patients with atypical or malignant meningiomas, adjuvant RT was associated with significantly improved PFS, and Ki-67 index >10% was associated with a trend toward worse PFS. Given the long-term survival, high recurrence rates, and efficacy of salvage therapy, patients with atypical and malignant meningiomas should be monitored systematically long after initial treatment.

Brachypodium Antifreeze Protein Gene Products Inhibit Ice Recrystallisation, Attenuate Ice Nucleation, and Reduce Immune Response.

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, Brachypodium AFP genes encode two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models which indicate that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins used to form ice at high sub-zero temperatures. These models are consistent with the experimentally demonstrated decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Additionally, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22). Structural models suggested that this was due to the affinity of the LRR domains to flg22. Overall, it is remarkable that the Brachypodium genes play multiple distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins' ability to adsorb to ice as well as to attenuate bacterial ice nucleation and the host immune response.

Optimization of flow path parameters for enhanced sensitivity lateral flow devices.

Lateral flow devices (LFDs) or lateral flow tests (LFTs) are one of the most widely used biosensor platforms for point-of-care (POC) diagnostics. The basic LFD design has remained largely unchanged since its first appearance, and this has limited LFD use in clinical applications due to a general lack of analytical sensitivity. We report here a comprehensive study of the use of laser-patterned geometric control barriers that influence the flow dynamics within an LFD, with the specific aim of enhancing LFD sensitivity and lowering the limit of detection (LOD). This control of sample flow produces an increase in the time available for optimizing the binding kinetics of the implemented assay. The geometric modification to the flow path is in the form of a constriction that is produced by depositing a photo-sensitive polymer onto the nitrocellulose membrane which when polymerized, creates impermeable barrier walls through the depth of the membrane. Both the position of the constriction within the flow path and the number of constrictions allow for an increase in the sensitivity because of a slower overall flow rate within the test and a larger volume of sample per unit width of the test line. For these high sensitivity LFDs (HS-LFD), through optimization of the constriction position and addition of a second constriction we attained a 62% increase in test line color intensity for the detection of procalcitonin (PCT) and were also able to lower the LOD from 10 ng/mL to 1 ng/mL. In addition, of relevance for future commercial exploitation, this also significantly decreases the antibody consumption per device leading to reduced costs for test production. We have further tested our HS-LFD with contrived human samples, validating its application for future clinical use.

Fully Masculinized 46,XX Individuals with Congenital Adrenal Hyperplasia: Perspective Regarding Sex of Rearing and Surgery.

Current guidelines for gender assignment for all 46,XX congenital adrenal hyperplasia (CAH) continue to be female. This decision is most challenging for individuals with a 46,XX karyotype born with (CAH) having severely masculinized genitalia (Prader 4 or 5). They may be at significant risk for quality of life (QoL) and psychological health. More outcome information currently exists for such individuals assigned male than female. Most available data for those raised females do not indicate the extent of masculinization at birth, so there are minimal outcome data to compare with those raised males. Gender dissatisfaction among those raised females may be related to the degree of prenatal androgen excess in the brain evidenced by external genital masculinization. Also, additional brain maturation after birth, especially during puberty, is impacted by postnatal androgen excess resulting from inadequate androgen suppression. The purpose of this perspective is to suggest that both female and male assignment be considered. Most who have been raised male at birth have positive adult outcomes. This consideration should occur after discussions with full disclosure to the parents. The lack of more outcome data highlights the need for further information. This perspective also suggests that surgery should be deferred whether assigned female or male at least until gender identity is apparent to preserve the potential for male sexual function and prevent irrevocable loss of sensitive erotic tissue. While the gender fluidity is recognized, it is important to consider potential subsequent need for gender reassignment and extent of masculinization, particularly at the time of gender determination.

Cheek Donor Site for Full-Thickness Skin Graft Repair of the Nasal Ala: Outcomes of a Retrospective Cohort Study.

BACKGROUND: Full-thickness skin grafts (FTSGs) are useful repairs for reconstructing nasal alar defects. Traditional donor sites include the preauricular, postauricular, and supraclavicular skin. OBJECTIVE: To evaluate esthetic outcomes and complications of nasal alar defects repaired with FTSGs from the medial cheek. MATERIALS AND METHODS: A retrospective chart review of Mohs surgery patients who had FTSG repair of the nasal ala between January 2015 and August 2020 was performed. Demographic, surgery, and follow-up visit data were reviewed. Cosmesis was rated by a facial plastic surgeon, a Mohs surgeon, and a plastic surgeon using baseline, defect, and follow-up visit photographs. RESULTS: Sixty-nine patients with FTSG repairs of nasal alar defects were identified. 51 of 69 patients (73.9%) had the cheek donor site, and 18 of 69 patients (26.1%) had a noncheek donor site. The mean (SD) rater visual analog score for both cohorts was good with no significant difference (cheek: 65.9 [13.8]; noncheek: 66.1 [15.3]; p = .96). A notable difference in the complication rate by donor site was observed (cheek: 6.9%, noncheek: 16.7%; p = .13), although it did not reach significance. CONCLUSION: The cheek is a reliable FTSG donor site for nasal alar defects after Mohs micrographic surgery, with a trend toward fewer complications.

MicroRNAs-A Promising Tool for Asthma Diagnosis and Severity Assessment: A Systematic Review.

Micro RNAs (miRNAs) are short, non-coding RNAs (Ribonucleic acids) with regulatory functions that could prove useful as biomarkers for asthma diagnosis and asthma severity-risk stratification. The objective of this systematic review is to identify panels of miRNAs that can be used to support asthma diagnosis and severity-risk assessment. Three databases (Medline, Embase, and SCOPUS) were searched up to 15 September 2020 to identify studies reporting differential expression of specific miRNAs in the tissues of adults and children with asthma. Studies reporting miRNAs associations in animal models that were also studied in humans were included in this review. We identified 75 studies that met our search criteria. Of these, 66 studies reported more than 200 miRNAs that are differentially expressed in asthma patients when compared to non-asthmatic controls. In addition, 16 studies reported 17 miRNAs that are differentially expressed with differences in asthma severity. We were able to construct two panels of miRNAs that are expressed in blood and can serve as core panels to further investigate the practicality and efficiency of using miRNAs as non-invasive biomarkers for asthma diagnosis and severity-risk assessment, respectively.

An investigation of kV mini-GRID spatially fractionated radiation therapy: dosimetry and preclinical trial.

Objective. To develop and characterize novel methods of extreme spatially fractionated kV radiation therapy (including mini-GRID therapy) and to evaluate efficacy in the context of a pre-clinical mouse study.Approach. Spatially fractionated GRIDs were precision-milled from 3 mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1 mm diameter beams, and three 'bar' GRIDs created 1 × 20 mm rectangular fields. GRIDs projected 20 × 20 mm2fields at isocenter, and beamlets were spaced at 1, 1.25, and 1.5 mm, respectively. Peak-to-valley ratios and dose distributions were evaluated with Gafchromic film. Syngeneic transplant tumors were induced by intramuscular injection of a soft tissue sarcoma cell line into the gastrocnemius muscle of C57BL/6 mice. Tumor-bearing mice were randomized to four groups: unirradiated control, conventional irradiation of entire tumor, GRID therapy, and hemi-irradiation (half-beam block, 50% tumor volume treated). All irradiated mice received a single fraction of 15 Gy.Results. High peak-to-valley ratios were achieved (bar GRIDs: 11.9 ± 0.9, 13.6 ± 0.4, 13.8 ± 0.5; pencil-beam GRIDs: 18.7 ± 0.6, 26.3 ± 1.5, 31.0 ± 3.3). Pencil-beam GRIDs could theoretically spare more intra-tumor immune cells than bar GRIDs, but they treat less tumor tissue (3%-4% versus 19%-23% area receiving 90% prescription, respectively). Bar GRID and hemi-irradiation treatments significantly delayed tumor growth (P < 0.05), but not as much as a conventional treatment (P < 0.001). No significant difference was found in tumor growth delay between GRID and hemi-irradiation.Significance. High peak-to-valley ratios were achieved with kV grids: two-to-five times higher than values reported in literature for MV grids. GRID irradiation and hemi-irradiation delayed tumor growth, but neither was as effective as conventional whole tumor uniform dose treatment. Single fraction GRID therapy could not initiate an anti-cancer immune response strong enough to match conventional RT outcomes, but follow-up studies will evaluate the combination of mini-GRID with immune checkpoint blockade.

Semi-quantitative detection of inflammatory biomarkers using a laser-patterned multiplexed lateral flow device.

Inflammatory markers including C-reactive protein (CRP) and procalcitonin (PCT) have been shown to be useful biomarkers to improve triage speed and prevent the inappropriate use of antibiotics for infections such as pneumonia. Here, we present a novel and exciting solution to guide the administration of antibiotic treatment via rapid, semi-quantitative and multiplexed detection of CRP and PCT using an advanced lateral flow device (LFD) designed to have multiple parallel flow-paths, produced via the precise laser-based partitioning of the single flow-path of a standard LFD. Each flow-path within this multiplexed LFD has a unique detection capability which permits tailored detection of CRP within a predefined cut-off range (20 µg/mL - 100 µg/mL) and PCT above a pre-defined threshold (0.5 ng/mL). We demonstrate the use of this LFD in the successful detection of CRP and PCT semi-quantitatively within spiked human serum samples. This multiplexed near-patient assay has potential for development into a rapid triage and treatment of patients with suspected pneumonia.

Robbing Peter to Pay Paul: Chlorhexidine gluconate demonstrates short-term efficacy and long-term cytotoxicity.

Wound cleansing agents are routine in wound care and preoperative preparation. Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove® and Dial® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with ~100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue. CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post-treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth. Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post-CHG application, likely serving as a reservoir to re-populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

Pseudoaneurysm following Two-Stage Hip Revision with Fasciotomy.

In the setting of total hip arthroplasty (THA), pseudoaneurysms are extremely rare and can be difficult to diagnose, as clinical symptoms can mimic symptoms of other more common complications, such as periprosthetic joint infection, hematoma, and nerve damage. We present a case of a 69-year-old male with a history of slipped capital femoral epiphysis 56 years prior and subsequent right THA. The right hip primary arthroplasty was subsequently complicated by multiple dislocations and recurrent prosthetic joint infections. The most recent infection was treated with debridement, antibiotics, and implant retention (DAIR) in 2017. The patient later presented in 2019 with right thigh pain. Upon further analysis, he was diagnosed with Streptococcus bovis positive periprosthetic joint infection. The patient underwent a two-stage revision of the hip using an antibiotic spacer. Two weeks following the second stage, he presented with a sudden onset of uncontrolled atrial fibrillation with rapid ventricular response and a low hemoglobin. The computed tomography scan revealed a large hematoma involving both the anterior and posterior thigh compartments with lab markers that were questionable for infection. An operation to remove the hematoma revealed no purulence, and a large pulsatile pseudoaneurysm on the posterolateral aspect at the mid femur was found. A sharp bone fragment was noted next to the pseudoaneurysm. The pseudoaneurysm was repaired by a vascular surgeon, and the bone fragment was removed. Following this procedure, the patient developed a subsequent periprosthetic joint infection requiring a double DAIR procedure six weeks following the pseudoaneurysm repair and is now on chronic antibiotic suppression. Orthopedic surgeons should be aware of the potential for pseudoaneurysm in the setting of total joint arthroplasty when treating a postsurgical hematoma of sudden onset.

Cold Acclimation in Brachypodium Is Accompanied by Changes in Above-Ground Bacterial and Fungal Communities.

Shifts in microbiota undoubtedly support host plants faced with abiotic stress, including low temperatures. Cold-resistant perennials prepare for freeze stress during a period of cold acclimation that can be mimicked by transfer from growing conditions to a reduced photoperiod and a temperature of 4 °C for 2-6 days. After cold acclimation, the model cereal, Brachypodium distachyon, was characterized using metagenomics supplemented with amplicon sequencing (16S ribosomal RNA gene fragments and an internal transcribed spacer region). The bacterial and fungal rhizosphere remained largely unchanged from that of non-acclimated plants. However, leaf samples representing bacterial and fungal communities of the endo- and phyllospheres significantly changed. For example, a plant-beneficial bacterium, Streptomyces sp. M2, increased more than 200-fold in relative abundance in cold-acclimated leaves, and this increase correlated with a striking decrease in the abundance of Pseudomonas syringae (from 8% to zero). This change is of consequence to the host, since P. syringae is a ubiquitous ice-nucleating phytopathogen responsible for devastating frost events in crops. We posit that a responsive above-ground bacterial and fungal community interacts with Brachypodium's low temperature and anti-pathogen signalling networks to help ensure survival in subsequent freeze events, underscoring the importance of inter-kingdom partnerships in the response to cold stress.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the αMß2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG, encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing.

As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of ∼100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 µl of reagent, our test only utilises less than half the quantity (10 µl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance.

In order to survive subzero temperatures, some plants undergo cold acclimation (CA) where low, nonfreezing temperatures, and/or shortened day lengths allow cold-hardening and survival during subsequent freeze events. Central to this response is the plasma membrane (PM), where low temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first PM proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time-course experiment investigated CA-induced changes in the proteome following two-phase partitioning PM enrichment and label-free quantification by nano-liquid chromatography-mass spectrophotometry. Two days of CA were sufficient for membrane protection as well as an initial increase in sugar levels and coincided with a significant change in the abundance of 154 proteins. Prolonged CA resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained CA response elicited over several days. A meta-analysis revealed that the identified PM proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress, and salt resistance suggesting crosstalk between stress responses, such that CA may prime plants for other abiotic and biotic stresses. The PM proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.