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CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

Background To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. Results Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. Conclusions Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections 1 – 3 . Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4 – 11 ), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication–transcription complex (RTC) 12 , 13 , in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27 , a robust early innate signature of SARS-CoV-2 (ref. 14 ), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae . Seronegative healthcare workers with an innate signature of infection preferentially expand pre-existing T cells targeting the conserved replication transcription complex of SARS-CoV-2 in abortive infection.

Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical β-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.

Background Physical punishment at home and in schools is widespread around the world. Systematic reviews and meta-analyses have synthesized evidence, mostly from high-income countries (HICs), showing that physical punishment relates to multiple detrimental individual outcomes. Yet, less work has been done to synthesize the evidence on the association between physical punishment at home and schools and child, adolescent, and adult outcomes in low- and middle-income countries (LMICs), where more than 90% of children live and physical punishment is most socially normative and prevalent. In this manuscript, we present a protocol for a systematic review and meta-analysis on the characteristics of the research, associations, and variation in associations, between physical punishment at home and in schools and child, adolescent, and adult outcomes in LMICs. Methods We will conduct a review of studies published in peer-reviewed journals using quantitative methods to assess the association between physical punishment in childhood and/or adolescence and individual outcomes in LMICs. We will search for studies in 10 different databases using keywords in English, Spanish, Portuguese, Arabic, and Chinese related to physical punishment. We will extract qualitative data from the studies and the statistics needed to transform all study-level effect sizes into standardized mean difference effect sizes. For the analyses, we will employ multi-level meta-analyses to use multiple effect sizes per study and leverage within-study variation as well as between study variation using moderation analysis. Besides the meta-analyses, we will also conduct a narrative synthesis of the findings. Discussion The proposed systematic review and meta-analysis will provide timely evidence to inform global research, policy, and practice on the links between physical punishment and lifelong individual outcomes. Systematic review registration PROSPERO CRD42022347346

For years, the Federal Farm Credit System has played by a set of home-brewed accounting standards that have masked bad lending and lax regulation. Loosely interpreted and poorly understood by some of the system's own officials, the rules have kept alive loans that accountants and commercial-bank regulators say other lenders would have been forced to give up as hopeless. They appear to have left the system with hundreds of millions of dollars of still-unrecognized losses and only a hazy notion of where its finances really stand. In calmer times, the system's tangled bookkeeping would be little more than grist for esoteric debates among accountants, and the mess could be straightened out with gradual tinkering. But the farm economy's collapse has suddenly turned subtle accounting anomalies into the difference between modest losses and catastrophic ones. Even though the Farm Credit System has asked for a federal bailout of its $74 billion loan portfolio, many accountants and bankers doubt that the system has yet to fully own up to its problems. Holder of more than one-third of the nation's staggering $212 billion of farm debt, the system is a crazy-quilt confederation of 12 regional banks, each with a land bank for land loans, an intermediate credit bank for planting loans and a bank for farm cooperatives. A 37th unit is a central bank for farm cooperatives. The system finances its loans by selling bonds and notes; it has about $70 billion worth outstanding. They aren't government-backed, though the system's regulator, the Farm Credit Administration, is a federal agency.

The General Accounting Office projected that the Farm Credit System will have a loss of $2.6 billion for the 12 months ending next June 30, and questioned the validity of some of the system's financial reports. James Roll, a senior vice president of the Farm Credit System funding arm, attacked the method used by the GAO to arrive at the projection, saying the method \"is open to serious question.\" He added that the system is updating its financial data and expects to have, before Oct. 31, its own projection of performance. The GAO review began three months ago at the request of several congressmen. The projections, extrapolations based on the system's June 30 financial data, assume a persisting farm slump, little change in the system's current method of establishing reserves for future losses and elevated funding costs. The system sells bonds to finance its loans; news of the system's woes have driven the spreads on those bonds to 80 to 100 basis points above comparable Treasury bills. A basis point is 1/100 percentage point.

Background The high prevalence of chronic diseases, including congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), and diabetes mellitus (DM), accounts for a large burden of cost and poor health outcomes in US hospitals, and home telehealth (HT) monitoring has been proposed to improve outcomes. Objective To measure the association between HT initiation and 12-month inpatient hospitalizations, emergency department (ED) visits, and mortality in veterans with CHF, COPD, or DM. Design Comparative effectiveness matched cohort study. Patients Veterans aged 65 years and older treated for CHF, COPD, or DM. Main Measures We matched veterans initiating HT with veterans with similar demographics who did not use HT (1:3). Our outcome measures included a 12-month risk of inpatient hospitalization, ED visits, and all-cause mortality. Key Results A total of 139,790 veterans with CHF, 65,966 with COPD, and 192,633 with DM were included in this study. In the year after HT initiation, the risk of hospitalization was not different in those with CHF (adjusted odds ratio [aOR] 1.01, 95% confidence interval [95%CI] 0.98–1.05) or DM (aOR 1.00, 95%CI 0.97–1.03), but it was higher in those with COPD (aOR 1.15, 95%CI 1.09–1.21). The risk of ED visits was higher among HT users with CHF (aOR 1.09, 95%CI 1.05–1.13), COPD (1.24, 95%CI 1.18–1.31), and DM (aOR 1.03, 95%CI 1.00–1.06). All-cause 12-month mortality was lower in those initiating HT monitoring with CHF (aOR 0.70, 95%CI 0.67–0.73) and DM (aOR 0.79, 95%CI 0.75–0.83), but higher in COPD (aOR 1.08, 95%CI 1.00–1.16). Conclusions The initiation of HT was associated with increased ED visits, no change in hospitalizations, and lower all-cause mortality in patients with CHF or DM, while those with COPD had both higher healthcare utilization and all-cause mortality.

Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 Å from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.