A modest increase in fire weather overcomes resistance to fire spread in recently burned boreal forests.

Recently burned boreal forests have lower aboveground fuel loads, generating a negative feedback to subsequent wildfires. Despite this feedback, short-interval reburns (≤20 years between fires) are possible under extreme weather conditions. Reburns have consequences for ecosystem recovery, leading to enduring vegetation change. In this study, we characterize the strength of the fire-fuel feedback in recently burned Canadian boreal forests and the weather conditions that overwhelm resistance to fire spread in recently burned areas. We used a dataset of daily fire spread for thousands of large boreal fires, interpolated from remotely sensed thermal anomalies to which we associated local weather from ERA5-Land for each day of a fire's duration. We classified days with >3 ha of fire growth as spread days and defined burned pixels overlapping a fire perimeter ≤20 years old as short-interval reburns. Results of a logistic regression showed that the odds of fire spread in recently burned areas were ~50% lower than in long-interval fires; however, all Canadian boreal ecozones experienced short-interval reburning (1981-2021), with over 100,000 ha reburning annually. As fire weather conditions intensify, the resistance to fire spread declines, allowing fire to spread in recently burned areas. The weather associated with short-interval fire spread days was more extreme than the conditions during long-interval spread, but overall differences were modest (e.g. relative humidity 2.6% lower). The frequency of fire weather conducive to short-interval fire spread has significantly increased in the western boreal forest due to climate warming and drying (1981-2021). Our results suggest an ongoing degradation of fire-fuel feedbacks, which is likely to continue with climatic warming and drying.

Harnessing noncanonical crRNA for highly efficient genome editing.

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

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.

An educational intervention for improving knowledge, attitude, and practice of dietary salt intake among individuals with hypertension in public sector secondary care facilities, Agra, India, 2021.

We conducted a pre-post intervention study to determine knowledge, attitude, and practice toward dietary salt intake before, immediately, and 1-month after nurse-led one-on-one counseling. We purposively selected three public health facilities in Agra, India, and enrolled all eligible hypertensive patients aged 18-60 under treatment for ≥6 months. Of the 153 patients at the 1-month follow-up, counseling improved knowledge (4% vs. 42%, p < .001), a greater prioritization of a low salt diet (34% vs. 52%, p < .001), and practice of adding less salt to the dough (48% to 41%, p < .001). The counseling intervention improved knowledge, attitude, and practice toward dietary salt intake.

New approaches to dissect leaf hydraulics reveal large gradients in living tissues of tomato leaves.

The water status of the living tissue in leaves is critical in determining plant function and global exchange of water and CO2. Despite significant advances in the past two decades, persistent questions remain about the tissue-specific origins of leaf hydraulic properties and their dependence on water status. We use a fluorescent nanoparticle reporter that provides water potential in the mesophyll apoplast adjacent to the epidermis of intact leaves to complement existing methods based on the Scholander Pressure Chamber (SPC). Working in tomato leaves, this approach provides access to the hydraulic conductance of the whole leaf, xylem, and outside-xylem tissues. These measurements show that, as stem water potential decreases, the water potential in the mesophyll apoplast can drop below that assessed with the SPC and can fall significantly below the turgor loss point of the leaf. We find that this drop in potential, dominated by the large loss (10-fold) of hydraulic conductance of the outside-xylem tissue, is not however strong enough to significantly limit transpiration. These observations highlight the need to reassess models of water transfer through the outside-xylem tissues, the potential importance of this tissue in regulating transpiration, and the power of new approaches for probing leaf hydraulics.

Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

Localized measurements of water potential reveal large loss of conductance in living tissues of maize leaves.

The water status of the living tissue in leaves between the xylem and stomata (outside xylem zone (OXZ) plays a critical role in plant function and global mass and energy balance but has remained largely inaccessible. We resolve the local water relations of OXZ tissue using a nanogel reporter of water potential (ψ), AquaDust, that enables an in situ, nondestructive measurement of both ψ of xylem and highly localized ψ at the terminus of transpiration in the OXZ. Working in maize (Zea mays L.), these localized measurements reveal gradients in the OXZ that are several folds larger than those based on conventional methods and values of ψ in the mesophyll apoplast well below the macroscopic turgor loss potential. We find a strong loss of hydraulic conductance in both the bundle sheath and the mesophyll with decreasing xylem potential but not with evaporative demand. Our measurements suggest the OXZ plays an active role in regulating the transpiration path, and our methods provide the means to study this phenomenon.

Programmable RNA detection with CRISPR-Cas12a.

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Broadleaf tree phenology and springtime wildfire occurrence in boreal Canada.

Although broadleaf tree species of the boreal biome have a lower flammability compared to conifers, there is a period following snow melt and prior to leaf flush (i.e., greenup), termed the "spring window" by fire managers, when these forests are relatively conducive to wildfire ignition and spread. The goal of this study was to characterize the duration, timing, and fire proneness of the spring window across boreal Canada and assess the link between these phenological variables and the incidence of springtime wildfires. We used remotely sensed snow cover and greenup data to identify the annual spring window for five boreal ecozones from 2001 to 2021 and then compared seasonality of wildfire starts (by cause) and fire-conducive weather in relation to this window, averaged over the 21-year period. We conducted a path analysis to concomitantly evaluate the influence of the spring window's duration, the timing of greenup, and fire-conducive weather on the annual number and the seasonality of spring wildfires. Results show that the characteristics of spring windows vary substantially from year to year and among geographic zones, with the interior west of Canada having the longest and most fire-conducive spread window and, accordingly, the greatest springtime wildfire activity. We also provide support for the belief that springtime weather generally promotes wind-driven, rather than drought-driven wildfires. The path analyses show idiosyncratic behavior among ecozones, but, in general, the seasonality of the wildfire season is mainly driven by the timing of the greenup, whereas the number of spring wildfires mostly responds to the duration of the spring window and the frequency of fire-conducive weather. The results of this study allows us to better understand and anticipate the biome-wide changes projected for the northern forests of North America.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential across many biological and medical disciplines, from understanding basic ecosystem processes and how organisms respond to environmental change, to disease diagnosis and detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta, and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than other real time-PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a lab setting, reduces the risk of cross-contamination, and can be completed in less than one hour. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

Post COVID-19 syndrome and new onset diseases: A prospective observational study.

Introduction: The National Institute of Health and Care Excellence (NICE) has defined the terms, 'acute coronavirus disease 2019' (COVID-19), 'ongoing symptomatic COVID-19' and 'post-COVID-19 syndrome', with the latter two described as having persistent symptoms after the onset of COVID-19 symptoms for 4-12 weeks and >12 weeks, respectively. Persistent symptoms can either be due to the after-effects of COVID-19 or new-onset diseases after acute COVID-19. All symptoms observed beyond 4 weeks after the onset of COVID-19 need not be present at the time of onset. Previous studies on persistent post-COVID-19 symptoms have not mentioned new-onset diseases after acute COVID-19, and only a select few studies have discussed such new-onset symptoms. Methods: Ninety-five patients who attended the post-COVID-19 clinic completed the requisite follow-up till 16 weeks after COVID-19 symptom onset. Data was recorded on a predesigned proforma. Necessary investigations were conducted to rule out any other cause of persistent symptoms. Results: Fatigue (62.1%), breathlessness (50.5%) and cough (27.4%) were the most common symptoms present beyond 4 weeks after the onset of COVID-19 symptoms. Forty-nine (51.57%) patients developed post-COVID-19 syndrome - their severity of symptoms (odds ratio [OR] 17.77) and longer duration of hospital stay (OR 1.095) during acute disease were significantly associated with the development of post-COVID-19 syndrome. During follow-up, 25 patients developed new-onset symptoms, such as diabetes mellitus, hypertension and idiopathic tachycardia. Conclusion: Patients can have persistent symptoms, new-onset symptoms and new-onset diseases after recovery from acute COVID-19.

Programmable RNA detection with CRISPR-Cas12a.

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or 'SAHARA' to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Programmable RNA detection with CRISPR-Cas12a.

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans- cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or 'SAHARA' to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg 2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

The Burden of Hypertension and Prehypertension in a Community Health Centre of Haryana.

Background Hypertension (HTN) is endemic in India and it is considered a public health challenge in both economically developed and developing nations. Unfortunately, despite its high prevalence, its awareness, treatment, and control status are low in urban as well as rural Indian populations. Objectives To determine the burden of hypertension and prehypertension in a Community Health Center (CHC) and to find the association of hypertension with the age group and sex of study subjects. Methodology A cross-sectional study was carried out among 713 patients of age 20 years and above attending the Out Patient Department (OPD) of the Community Health Centre (CHC), Barwala in the Hisar district of Haryana. JNC 7 classification of blood pressure was used to diagnose hypertension. The collected data was analysed using the Statistical Package for Social Sciences (SPSS) version 20.0. Appropriate statistical tests were used. Result Out of the total patients enrolled, 200 (28.1%) were found to be hypertensives. The burden of prehypertension was further observed to be 28.1%. About (61.6%) of OPD patients were female. Among hypertensive patients, nearly half (48.8%) were in the age group of 60-80 years whereas the majority of the hypertensives (56%) were females. Conclusion In our study, more than 50 percent of OPD patients were found to have hypertension and pre-hypertension. Health-seeking behavior was more among females.

Experimental testing of solar-based air heater roughed with discrete V-down rib and staggered element.

A rough rectangular channel of solar-based air heater (SBAH) is made and tested. The work consists of an effort to perceive the proportion of heat discharge and frictional behavior of air passing over a roughened rectangular channel. The absorber surface of SBAH is roughed with discrete V-down rib and staggered element roughness having different values of relative rib pitch (P/e) that ranged from 6 to 14. Fixed parameters such as relative gap size (g/e), relative staggered element pitch (P'/P), numeral of gaps (Ng), relative staggered element size (r/g), and relative rib height (e/D) are considered 4, 0.4, 3,1, and 0.0433, respectively, all throughout the study. The flow Reynolds number (Re) changes from 4000 to 14,000; consequently the Nusselt number (Nu) and friction factor (f) reach up to 2.16 and 2.73 times, respectively, with respect to plane surface. The optimum rise in terms of thermal-hydraulic performance (THP) is gained analogous to a P/e of 10. The correlation for heat transfer function, R(e+), and roughness function, G(e+), is given to anticipate the performance of roughness.

Ancestral origins are associated with SARS-CoV-2 susceptibility and protection in a Florida patient population.

COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1x10-5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio[OR] = 0.173; 95% confidence interval[CI], 0.08-0.36 for G allele; p = 5.15× 10-5 and index rs768632395: OR = 0.166; 95% CI, 0.07-0.35 for A allele; p = 4.25×10-6, respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.

A signal-like role for floral humidity in a nocturnal pollination system.

Previous studies have considered floral humidity to be an inadvertent consequence of nectar evaporation, which could be exploited as a cue by nectar-seeking pollinators. By contrast, our interdisciplinary study of a night-blooming flower, Datura wrightii, and its hawkmoth pollinator, Manduca sexta, reveals that floral relative humidity acts as a mutually beneficial signal in this system. The distinction between cue- and signal-based functions is illustrated by three experimental findings. First, floral humidity gradients in Datura are nearly ten-fold greater than those reported for other species, and result from active (stomatal conductance) rather than passive (nectar evaporation) processes. These humidity gradients are sustained in the face of wind and are reconstituted within seconds of moth visitation, implying substantial physiological costs to these desert plants. Second, the water balance costs in Datura are compensated through increased visitation by Manduca moths, with concomitant increases in pollen export. We show that moths are innately attracted to humid flowers, even when floral humidity and nectar rewards are experimentally decoupled. Moreover, moths can track minute changes in humidity via antennal hygrosensory sensilla but fail to do so when these sensilla are experimentally occluded. Third, their preference for humid flowers benefits hawkmoths by reducing the energetic costs of flower handling during nectar foraging. Taken together, these findings suggest that floral humidity may function as a signal mediating the final stages of floral choice by hawkmoths, complementing the attractive functions of visual and olfactory signals beyond the floral threshold in this nocturnal plant-pollinator system.