PAM-free diagnostics with diverse type V CRISPR-Cas systems.

Type V CRISPR-Cas effectors have revolutionized molecular diagnostics by facilitating the detection of nucleic acid biomarkers. However, their dependence on the presence of protospacer adjacent motif (PAM) sites on the target double-stranded DNA (dsDNA) greatly limits their flexibility as diagnostic tools. Here we present a novel method named PICNIC that solves the PAM problem for CRISPR-based diagnostics with just a simple ∼10-min modification to contemporary CRISPR-detection protocols. Our method involves the separation of dsDNA into individual single-stranded DNA (ssDNA) strands through a high- temperature and high-pH treatment. We then detect the released ssDNA strands with diverse Cas12 enzymes in a PAM-free manner. We show the utility of PICNIC by successfully applying it for PAM-free detection with three different subtypes of the Cas12 family- Cas12a, Cas12b, and Cas12i. Notably, by combining PICNIC with a truncated 15-nucleotide spacer containing crRNA, we demonstrate PAM-independent detection of clinically important single- nucleotide polymorphisms with CRISPR. We apply this approach to detect the presence of a drug-resistant variant of HIV-1, specifically the K103N mutant, that lacks a PAM site in the vicinity of the mutation. Additionally, we successfully translate our approach to clinical samples by detecting and genotyping HCV-1a and HCV-1b variants with 100% specificity at a PAM-less site within the HCV genome. In summary, PICNIC is a simple yet groundbreaking method that enhances the flexibility and precision of CRISPR-Cas12-based diagnostics by eliminating the restriction of the PAM sequence.

A novel method to quantify fibrin-fibrin and fibrin-α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma.

BACKGROUND: The widespread use of the antifibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, factor (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis. OBJECTIVES: Here, we developed a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. METHODS: Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α2AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed. RESULTS: The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05). CONCLUSION: We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach, we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.

STN7 is not essential for developmental acclimation of Arabidopsis to light intensity.

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions.

Discovery of plasma proteome markers associated with clinical outcome and immunological stress after cardiac surgery.

Background: Molecular mechanisms underlying perioperative acute phase reactions in cardiac surgery are largely unknown. We aimed to characterise perioperative alterations of the acute phase plasma proteome in a cohort of adult patients undergoing on-pump cardiac surgery using high-throughput mass spectrometry and to identify candidate proteins potentially relevant to postoperative clinical outcome through a novel, multi-step approach. Methods: This study is an analysis of the Bern Perioperative Biobank, a prospective cohort of adults who underwent cardiac surgery with the use of cardiopulmonary bypass (CPB) at Bern University Hospital between January and December 2019. Blood samples were taken before induction of anaesthesia and on postoperative day one. Proteomic analyses were performed by mass spectrometry. Through a multi-step, exploratory approach, hit-proteins were first identified according to their perioperative prevalence and dynamics. The set of hit-proteins were associated with predefined clinical outcome measures (all-cause one-year mortality, length of hospital stay, postoperative myocardial infarction and stroke until hospital discharge). Results: 192 patients [75.5% male, median age 67.0 (IQR 60.0-73.0)] undergoing cardiac surgery with the use of CPB were included in this analysis. In total, we identified and quantified 402 proteins across all samples, whereof 30/402 (7%) proteins were identified as hit-proteins. Three hit-proteins-LDHB, VCAM1 and IGFBP2-demonstrated the strongest associations with clinical outcomes. After adjustment both for age, sex, BMI and for multiple comparisons, the scaled preoperative levels of IGFBP2 were associated with 1-year all-cause mortality (OR 10.63; 95% CI: 2.93-64.00; p = 0.046). Additionally, scaled preoperative levels of LDHB (OR 5.58; 95% CI: 2.58-8.57; p = 0.009) and VCAM1 (OR 2.32; 95% CI: 0.88-3.77; p = 0.05) were found to be associated with length of hospital stay. Conclusions: We identified a subset of promising candidate plasma proteins relevant to outcome after on-pump cardiac surgery. IGFBP2 showed a strong association with clinical outcome measures and a significant association of preoperative levels with 1-year all-cause mortality. Other proteins strongly associated with outcome were LDHB and VCAM1, reflecting the dynamics in the acute phase response, inflammation and myocardial injury. We recommend further investigation of these proteins as potential outcome markers after cardiac surgery. Clinical Trial Registration: ClinicalTrials.gov; NCT04767685, data are available via ProteomeXchange with identifier PXD046496.

Comparative proteomics of thylakoids from Arabidopsis grown in laboratory and field conditions.

Compared to controlled laboratory conditions, plant growth in the field is rarely optimal since it is frequently challenged by large fluctuations in light and temperature which lower the efficiency of photosynthesis and lead to photo-oxidative stress. Plants grown under natural conditions therefore place an increased onus on the regulatory mechanisms that protect and repair the delicate photosynthetic machinery. Yet, the exact changes in thylakoid proteome composition which allow plants to acclimate to the natural environment remain largely unexplored. Here, we use quantitative label-free proteomics to demonstrate that field-grown Arabidopsis plants incorporate aspects of both the low and high light acclimation strategies previously observed in laboratory-grown plants. Field plants showed increases in the relative abundance of ATP synthase, cytochrome b 6 f, ferredoxin-NADP+ reductases (FNR1 and FNR2) and their membrane tethers TIC62 and TROL, thylakoid architecture proteins CURT1A, CURT1B, RIQ1, and RIQ2, the minor monomeric antenna complex CP29.3, rapidly-relaxing non-photochemical quenching (qE)-related proteins PSBS and VDE, the photosystem II (PSII) repair machinery and the cyclic electron transfer complexes NDH, PGRL1B, and PGR5, in addition to decreases in the amounts of LHCII trimers composed of LHCB1.1, LHCB1.2, LHCB1.4, and LHCB2 proteins and CP29.2, all features typical of a laboratory high light acclimation response. Conversely, field plants also showed increases in the abundance of light harvesting proteins LHCB1.3 and CP29.1, zeaxanthin epoxidase (ZEP) and the slowly-relaxing non-photochemical quenching (qI)-related protein LCNP, changes previously associated with a laboratory low light acclimation response. Field plants also showed distinct changes to the proteome including the appearance of stress-related proteins ELIP1 and ELIP2 and changes to proteins that are largely invariant under laboratory conditions such as state transition related proteins STN7 and TAP38. We discuss the significance of these alterations in the thylakoid proteome considering the unique set of challenges faced by plants growing under natural conditions.

Hospital-Acquired Bloodstream Infections in Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 Infection (Coronavirus Disease 2019): Association With Immunosuppressive Therapies.

BACKGROUND: Immunosuppressive therapies proposed for Coronavirus disease 2019 (COVID-19) management may predispose to secondary infections. We evaluated the association of immunosuppressive therapies with bloodstream-infections (BSIs) in hospitalized COVID-19 patients. METHODS: This was an institutional review board-approved retrospective, multicenter, cohort study of adults hospitalized with COVID-19 over a 5-month period. We obtained clinical, microbiologic and laboratory data from electronic medical records. Propensity-score-matching helped create balanced exposure groups. Demographic characteristics were compared across outcome groups (BSI/no BSI) using two-sample t-test and Chi-Square test for continuous and categorical variables respectively, while immunosuppressive therapy use was compared using McNemar's test. Conditional logistic regression helped assess the association between immunosuppressive therapies and BSIs. RESULTS: 13,007 patients were originally included, with propensity-score-matching producing a sample of 6,520 patients. 3.74% and 3.97% were diagnosed with clinically significant BSIs in the original and propensity-score-matched populations respectively. COVID-19 patients with BSIs had significantly longer hospitalizations, higher intensive care unit admission and mortality rates compared to those without BSIs. On univariable analysis, combinations of corticosteroids/anakinra [odds-ratio (OR) 2.00, 95% confidence intervals (C.I.) 1.05-3.80, P value.0342] and corticosteroids/tocilizumab [OR 2.13, 95% C.I. 1.16-3.94, P value .0155] were significantly associated with BSIs. On multivariable analysis (adjusting for confounders), combination corticosteroids/tocilizumab were significantly associated with any BSI [OR 1.97, 95% C.I. 1.04-3.73, P value.0386] and with bacterial BSIs [OR 2.13, 95% C.I. 1.12-4.05, p-value 0.0217]. CONCLUSIONS: Combination immunosuppressive therapies were significantly associated with BSI occurrence in COVID-19 patients; their use warrants increased BSI surveillance. Further studies are needed to establish their causative role.

Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb 3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni.

Bacterial C-type haem-copper oxidases in the cbb 3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb 3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCu A C (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c-1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.

A comparison of COVID-19 inpatients by HIV status.

COVID-19 in-hospital morbidity and mortality in people living with HIV (PLWH) were compared to HIV-negative COVID-19 patients within a New York City metropolitan health system, the hardest hit region in the United States early in the pandemic. A total of 10,202 inpatients were diagnosed with COVID-19, of which 99 were PLWH. PLWH were younger (58.3 years (SD = 12.42) versus 64.32 years (SD = 16.77), p < 0.001) and had a higher prevalence of men (73.7% versus 57.9%, p = 0.002) and Blacks (43.4% versus 21.7%, p < 0.001) than the HIV-negative population. PLWH had a higher prevalence of malignancies (18% versus 7%, p = < 0.001), chronic liver disease (12% versus 3%, p < 0.001), and end-stage renal disease (11% versus 4%, p = 0.007). Use of a ventilator, admission to the ICU, and in-hospital mortality were not different. Of the 99 PLWH, 12 were virally unsuppressed and 9 had CD4% < 14. Two of the 12 virally unsuppressed patients and 4/9 patients with CD4% < 14 died. Ninety-one of the 99 PLWH were on treatment for HIV, and 5 of the 8 not on treatment died. Among PLWH with prior values, absolute CD4 count decreased an average of 192 cells/mm3 at the time of COVID-19 diagnosis (p < 0.001). Hospitalized patients with HIV and COVID-19 coinfection did not have worse outcomes than the general population. Among PLWH, those with CD4%<14 or not on treatment for HIV had higher mortality rates. Those PLWH who received IL-6 inhibitors had lower mortality rates. PLWH given antifungal medications, hydroxychloroquine, antibiotics (including azithromycin), steroids, and vasopressors had higher mortality rates.

Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis.

Photosynthetic acclimation, the ability to adjust the composition of the thylakoid membrane to optimise the efficiency of electron transfer to the prevailing light conditions, is crucial to plant fitness in the field. While much is known about photosynthetic acclimation in Arabidopsis, to date there has been no study that combines both quantitative label-free proteomics and photosynthetic analysis by gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy. Using these methods we investigated how the levels of 402 thylakoid proteins, including many regulatory proteins not previously quantified, varied upon long-term (weeks) acclimation of Arabidopsis to low (LL), moderate (ML) and high (HL) growth light intensity and correlated these with key photosynthetic parameters. We show that changes in the relative abundance of cytb6 f, ATP synthase, FNR2, TIC62 and PGR6 positively correlate with changes in estimated PSII electron transfer rate and CO2 assimilation. Improved photosynthetic capacity in HL grown plants is paralleled by increased cyclic electron transport, which positively correlated with NDH, PGRL1, FNR1, FNR2 and TIC62, although not PGR5 abundance. The photoprotective acclimation strategy was also contrasting, with LL plants favouring slowly reversible non-photochemical quenching (qI), which positively correlated with LCNP, while HL plants favoured rapidly reversible quenching (qE), which positively correlated with PSBS. The long-term adjustment of thylakoid membrane grana diameter positively correlated with LHCII levels, while grana stacking negatively correlated with CURT1 and RIQ protein abundance. The data provide insights into how Arabidopsis tunes photosynthetic electron transfer and its regulation during developmental acclimation to light intensity.

A miRNA-145/TGF-ß1 negative feedback loop regulates the cancer-associated fibroblast phenotype.

The dissemination of cancer cells to local and distant sites depends on a complex and poorly understood interplay between malignant cells and the cellular and non-cellular components surrounding them, collectively termed the tumour microenvironment. One of the most abundant cell types of the tumour microenvironment is the fibroblast, which becomes corrupted by locally derived cues such as TGF-ß1 and acquires an altered, heterogeneous phenotype (cancer-associated fibroblasts, CAF) supportive of tumour cell invasion and metastasis. Efforts to develop new treatments targeting the tumour mesenchyme are hampered by a poor understanding of the mechanisms underlying the development of CAF. Here, we examine the contribution of microRNA to the development of experimentally-derived CAF and correlate this with changes observed in CAF derived from tumours. Exposure of primary normal human fibroblasts to TGF-ß1 resulted in the acquisition of a myofibroblastic CAF-like phenotype. This was associated with increased expression of miR-145, a miRNA predicted in silico to target multiple components of the TGF-ß signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-ß1-induced myofibroblastic differentiation and reverted CAF towards a normal fibroblast phenotype. We conclude that miR-145 is a key regulator of the CAF phenotype, acting in a negative feedback loop to dampen acquisition of myofibroblastic traits, a key feature of CAF associated with poor disease outcome.