A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis.

RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

mRNA cleavage by 21-nucleotide phasiRNAs determines temperature-sensitive male sterility in rice.

Temperature-sensitive male sterility is one of the core components for hybrid rice (Oryza sativa) breeding based on the 2-line system. We previously found that knockout of ARGONAUTE 1d (AGO1d) causes temperature-sensitive male sterility in rice by influencing phased small interfering RNA (phasiRNA) biogenesis and function. However, the specific phasiRNAs and their targets underlying the temperature-sensitive male sterility in the ago1d mutant remain unknown. Here, we demonstrate that the ago1d mutant displays normal female fertility but complete male sterility at low temperature. Through a multiomics analysis of small RNA (sRNA), degradome, and transcriptome, we found that 21-nt phasiRNAs account for the greatest proportion of the 21-nt sRNA species in rice anthers and are sensitive to low temperature and markedly downregulated in the ago1d mutant. Moreover, we found that 21-nt phasiRNAs are essential for the mRNA cleavage of a set of fertility- and cold tolerance-associated genes, such as Earlier Degraded Tapetum 1 (EDT1), Tapetum Degeneration Retardation (TDR), OsPCF5, and OsTCP21, directly or indirectly determined by AGO1d-mediated gene silencing. The loss of function of 21-nt phasiRNAs can result in upregulation of their targets and causes varying degrees of defects in male fertility and grain setting. Our results highlight the essential functions of 21-nt phasiRNAs in temperature-sensitive male sterility in rice and suggest their promising application in 2-line hybrid rice breeding in the future.

A cascade of bHLH-regulated pathways programs maize anther development.

The spatiotemporal development of somatic tissues of the anther lobe is necessary for successful fertile pollen production. This process is mediated by many transcription factors acting through complex, multi-layered networks. Here, our analysis of functional knockout mutants of interacting basic helix-loop-helix genes Ms23, Ms32, basic helix-loop-helix 122 (bHLH122), and bHLH51 in maize (Zea mays) established that male fertility requires all four genes, expressed sequentially in the tapetum (TP). Not only do they regulate each other, but also they encode proteins that form heterodimers that act collaboratively to guide many cellular processes at specific developmental stages. MS23 is confirmed to be the master factor, as the ms23 mutant showed the earliest developmental defect, cytologically visible in the TP, with the most drastic alterations in premeiotic gene expression observed in ms23 anthers. Notably, the male-sterile ms23, ms32, and bhlh122-1 mutants lack 24-nt phased secondary small interfering RNAs (phasiRNAs) and the precursor transcripts from the corresponding 24-PHAS loci, while the bhlh51-1 mutant has wild-type levels of both precursors and small RNA products. Multiple lines of evidence suggest that 24-nt phasiRNA biogenesis primarily occurs downstream of MS23 and MS32, both of which directly activate Dcl5 and are required for most 24-PHAS transcription, with bHLH122 playing a distinct role in 24-PHAS transcription.

Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci.

The anther-enriched phased, small interfering RNAs (phasiRNAs) play vital roles in sustaining male fertility in grass species. Their long non-coding precursors are synthesized by RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21- or 24-nucleotide phasiRNA loci (referred to as 21- or 24-PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate any PHAS locus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21- or 24-PHAS loci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants provided insights into TF timing with regard to phasiRNA biogenesis, including antagonistic roles for OUTER CELL LAYER4 and MALE STERILE23. Trans-activation assays in maize protoplasts of individual TFs using bulk-protoplast RNA-sequencing showed that two of the TFs coexpressed with 21-PHAS loci could activate several 21-nucleotide phasiRNA pathway genes but not transcription of 21-PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24-PHAS loci using single-cell (protoplast) RNA-sequencing, did not detect reproducible activation of either 21-PHAS or 24-PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci.

Diagnostic efficacy of an optimized nucleotide MALDI-TOF-MS assay for anti-tuberculosis drug resistance detection.

PURPOSE: We aimed at evaluating the diagnostic efficacy of a nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay to detect drug resistance of Mycobacterium tuberculosis. METHODS: Overall, 263 M. tuberculosis clinical isolates were selected to evaluate the performance of nucleic MALDI-TOF-MS for rifampin (RIF), isoniazid (INH), ethambutol (EMB), moxifloxacin (MXF), streptomycin (SM), and pyrazinamide (PZA) resistance detection. The results for RIF, INH, EMB, and MXF were compared with phenotypic microbroth dilution drug susceptibility testing (DST) and whole-genome sequencing (WGS), and the results for SM and PZA were compared with those obtained by WGS. RESULTS: Using DST as the gold standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 98.2%, 98.7%, and 0.97 for RIF; 92.8%, 99%, and 0.90 for INH; 82.4%, 98.0%, and 0.82 for EMB; and 92.6%, 99.5%, and 0.94 for MXF, respectively. Compared with WGS as the reference standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 97.4%, 100.0%, and 0.98 for RIF; 98.7%, 92.9%, and 0.92 for INH; 96.3%, 100.0%, and 0.98 for EMB; 98.1%, 100.0%, and 0.99 for MXF; 98.0%, 100.0%, and 0.98 for SM; and 50.0%, 100.0%, and 0.65 for PZA. CONCLUSION: The nucleotide MALDI-TOF-MS assay yielded highly consistent results compared to DST and WGS, suggesting that it is a promising tool for the rapid detection of sensitivity to RIF, INH, EMB, and MXF.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Mechanisms of ferroptosis with immune infiltration and inflammatory response in rotator cuff injury.

The processes driving ferroptosis and rotator cuff (RC) inflammation are yet unknown. The mechanism of ferroptosis and inflammation involved in the development of RC tears was investigated. The Gene Expression Omnibus database was used to obtain the microarray data relevant to the RC tears for further investigation. In this study, we created an RC tears rat model for in vivo experimental validation. For the additional function enrichment analysis, 10 hub ferroptosis-related genes were chosen to construct the correlation regulation network. In RC tears, it was discovered that genes related to hub ferroptosis and hub inflammatory response were strongly correlated. The outcomes of in vivo tests showed that RC tears were related to Cd68-Cxcl13, Acsl4-Sat1, Acsl3-Eno3, Acsl3-Ccr7, and Ccr7-Eno3 pairings in regulating ferroptosis and inflammatory response. Thus, our results show an association between ferroptosis and inflammation, providing a new avenue to explore the clinical treatment of RC tears.

Revisiting matrix hydrogel composed of gelatin and hyaluronic acid and its application in cartilage regeneration.

With the increasing incidence of knee osteoarthritis (KOA), the reparation of cartilage defects is gaining more attention. Given that tissue integration plays a critical role in repairing cartilage defects, tissue adhesive hydrogels are highly needed in clinics. We constructed a biomacromolecule-based bioadhesive matrix hydrogel and applied it to promote cartilage regeneration. The hydrogel was composed of methacrylate gelatin and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitroso) butyl amide modified hyaluronic acid (HANB). The methacrylate gelatin provided a stable hydrogel network as a scaffold, and the HANB served as a tissue-adhesive agent and could be favorable for the chondrogenesis of stem cells. Additionally, the chemically modified HA increased the swelling ratio and compressive modulus of the hydrogels. The results of our in vitro study revealed that the hydrogel was compatible with bone marrow stromal cells. In vivo, the hyaluronic-acid-containing hydrogels were found to promote articular cartilage regeneration in the defect site. Therefore, this biomaterial provides promising potential for cartilage repair.

The evolutionary history of small RNAs in Solanaceae.

The Solanaceae or "nightshade" family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family's small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.

PhasiRNAs in Plants: Their Biogenesis, Genic Sources, and Roles in Stress Responses, Development, and Reproduction.

Phased secondary small interfering RNAs (phasiRNAs) constitute a major category of small RNAs in plants, but most of their functions are still poorly defined. Some phasiRNAs, known as trans-acting siRNAs, are known to target complementary mRNAs for degradation and to function in development. However, the targets or biological roles of other phasiRNAs remain speculative. New insights into phasiRNA biogenesis, their conservation, and their variation across the flowering plants continue to emerge due to the increased availability of plant genomic sequences, deeper and more sophisticated sequencing approaches, and improvements in computational biology and biochemical/molecular/genetic analyses. In this review, we survey recent progress in phasiRNA biology, with a particular focus on two classes associated with male reproduction: 21-nucleotide (accumulate early in anther ontogeny) and 24-nucloetide (produced in somatic cells during meiosis) phasiRNAs. We describe phasiRNA biogenesis, function, and evolution and define the unanswered questions that represent topics for future research.

A spur to molecular geometry optimization: Gradient-enhanced universal kriging with on-the-fly adaptive ab initio prior mean functions in curvilinear coordinates.

We present a molecular geometry optimization algorithm based on the gradient-enhanced universal kriging (GEUK) formalism with ab initio prior mean functions, which incorporates prior physical knowledge to surrogate-based optimization. In this formalism, we have demonstrated the advantage of allowing the prior mean functions to be adaptive during geometry optimization over a pre-fixed choice of prior functions. Our implementation is general and flexible in two senses. First, the optimizations on the surrogate surface can be in both Cartesian coordinates and curvilinear coordinates. We explore four representative curvilinear coordinates in this work, including the redundant Coulombic coordinates, the redundant internal coordinates, the non-redundant delocalized internal coordinates, and the non-redundant hybrid delocalized internal Z-matrix coordinates. We show that our GEUK optimizer accelerates geometry optimization as compared to conventional non-surrogate-based optimizers in internal coordinates. We further showcase the power of the GEUK with on-the-fly adaptive priors for efficient optimizations of challenging molecules (Criegee intermediates) with a high-accuracy electronic structure method (the coupled-cluster method). Second, we present the usage of internal coordinates under the complete curvilinear scheme. A complete curvilinear scheme performs both surrogate potential-energy surface (PES) fitting and structure optimization entirely in the curvilinear coordinates. Our benchmark indicates that the complete curvilinear scheme significantly reduces the cost of structure minimization on the surrogate compared to the incomplete curvilinear scheme, which fits the surrogate PES in curvilinear coordinates partially and optimizes a structure in Cartesian coordinates through curvilinear coordinates via the chain rule.

Exploring torsional conformer space with physical prior mean function-driven meta-Gaussian processes.

We present a novel approach for systematically exploring the conformational space of small molecules with multiple internal torsions. Identifying unique conformers through a systematic conformational search is important for obtaining accurate thermodynamic functions (e.g., free energy), encompassing contributions from the ensemble of all local minima. Traditional geometry optimizers focus on one structure at a time, lacking transferability from the local potential-energy surface (PES) around a specific minimum to optimize other conformers. In this work, we introduce a physics-driven meta-Gaussian processes (meta-GPs) method that not only enables efficient exploration of target PES for locating local minima but, critically, incorporates physical surrogates that can be applied universally across the optimization of all conformers of the same molecule. Meta-GPs construct surrogate PESs based on the optimization history of prior conformers, dynamically selecting the most suitable prior mean function (representing prior knowledge in Bayesian learning) as a function of the optimization progress. We systematically benchmarked the performance of multiple GP variants for brute-force conformational search of amino acids. Our findings highlight the superior performance of meta-GPs in terms of efficiency, comprehensiveness of conformer discovery, and the distribution of conformers compared to conventional non-surrogate optimizers and other non-meta-GPs. Furthermore, we demonstrate that by concurrently optimizing, training GPs on the fly, and learning PESs, meta-GPs exhibit the capacity to generate high-quality PESs in the torsional space without extensive training data. This represents a promising avenue for physics-based transfer learning via meta-GPs with adaptive priors in exploring torsional conformer space.

Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.

Composite plants containing transgenic hairy roots produced with Agrobacterium rhizogenes-mediated transformation have become an important method to study the interaction between plants and arbuscular mycorrhizal fungi (AMF). Not all hairy roots induced by A. rhizogenes are transgenic, however, which leads to requirement of a binary vector to carry a reporter gene to distinguish transgenic roots from non-transformed hairy roots. The beta-glucuronidase gene (GUS) and fluorescent protein gene often are used as reporter markers in the process of hairy root transformation, but they require expensive chemical reagents or imaging equipment. Alternatively, AtMYB75, an R2R3 MYB transcription factor from Arabidopsis thaliana, recently has been used as a reporter gene in hairy root transformation in some leguminous plants and can cause anthocyanin accumulation in transgenic hairy roots. Whether AtMYB75 can be used as a reporter gene in the hairy roots of tomato and if the anthocyanins accumulating in the roots will affect AMF colonization, however, are still unknown. In this study, the one-step cutting method was used for tomato hairy root transformation by A.rhizogenes. It is faster and has a higher transformation efficiency than the conventional method. AtMYB75 was used as a reporter gene in tomato hairy root transformation. The results showed that the overexpression of AtMYB75 caused anthocyanin accumulation in the transformed hairy roots. Anthocyanin accumulation in the transgenic hairy roots did not affect their colonization by the arbuscular mycorrhizal fungus, Funneliformis mosseae strain BGC NM04A, and there was no difference in the expression of the AMF colonization marker gene SlPT4 in AtMYB75 transgenic roots and wild-type roots. Hence, AtMYB75 can be used as a reporter gene in tomato hairy root transformation and in the study of symbiosis between tomato and AMF.

Large Pressure Effects Caused by Internal Rotation in the s-cis-syn-Acrolein Stabilized Criegee Intermediate at Tropospheric Temperature and Pressure.

Criegee intermediates are important atmospheric oxidants, and quantitative kinetics for stabilized Criegee intermediates are key parameters for atmospheric modeling but are still limited. Here we report barriers and rate constants for unimolecular reactions of s-cis-syn-acrolein oxide (scsAO), in which the vinyl group makes it a prototype for Criegee intermediates produced in the ozonolysis of isoprene. We find that the MN15-L and M06-2X density functionals have CCSD(T)/CBS accuracy for the unimolecular cyclization and stereoisomerization of scsAO. We calculated high-pressure-limit rate constants by the dual-level strategy that combines (a) high-level wave function-based conventional transition-state theory (which includes coupled-cluster calculations with quasiperturbative inclusion of quadruple excitations because of the strongly multiconfigurational character of the electronic wave function) and (b) canonical variational transition-state theory with small-curvature tunneling based on a validated density functional. We calculated pressure-dependent rate constants both by system-specific quantum Rice-Ramsperger-Kassel theory and by solving the master equation. We report rate constants for unimolecular reactions of scsAO over the full range of atmospheric temperature and pressure. We found that the unimolecular reaction rates of this larger-than-previously studied Criegee intermediate depend significantly on pressure. Particularly, we found that falloff effects decrease the effective unimolecular cyclization rate constant of scsAO by about a factor of 3, but the unimolecular reaction is still the dominant atmospheric sink for scsAO at low altitudes. The large falloff caused by the inclusion of the stereoisomerization channel in the master equation calculations has broad implications for mechanistic analysis of reactions with competitive internal rotations that can produce stable rotamers.

24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers.

In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection, we analyzed tapetal cells, meiocytes and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. By integrating RNA and small RNA profiling plus single-molecule and small RNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum. Our data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.

Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ.

Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize (Zea mays), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus Ustilago maydis, to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zmmac1-1 anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses.

Should corticosteroids be administered for local infiltration analgesia in knee arthroplasty? A meta-analysis and systematic review.

WHAT IS KNOWN AND OBJECTIVE: The benefits of local infiltration analgesia (LIA) in knee arthroplasty (KA) have been well-documented. However, it is unknown whether adding a corticosteroid to the composition of the LIA is beneficial. This study aimed to investigate the efficacy and safety of administering periarticular steroids intraoperatively in patients who underwent KA through a systematic review and meta-analysis. METHODS: A systematic search was conducted to identify relevant randomized controlled trials in the PubMed, Embase, Web of Science and Cochrane databases up to January 19th, 2021 to perform a meta-analysis. Outcome variables included pain scores, total opioid consumption, knee range of motion (ROM) and postoperative complications. RESULTS: Corticosteroid injections did not reduce pain scores at 6, 12, 24 or 72 h postoperatively, although a minimal degree of transient pain relief was achieved at 48 h postoperatively compared with those in the placebo group, nor was there a significant difference in total opioid consumption. However, patients receiving corticosteroids did exhibit a transient ROM increase on postoperative days 1, 2 and 3. Since the minimal clinically important difference (MCID) for ROM is unclear, it is unknown if the improvement in ROM is clinically significant. WHAT IS NEW AND CONCLUSION: Our specific end-point analysis demonstrated that corticosteroid administration did not provide pain relief or reduce opioid consumption compared with placebo. However, corticosteroids might provide a statistically significant, though transient and minimal improvement in knee ROM after KA, although no firm conclusions about the benefits of administering corticosteroids in KA can be made based on the available evidence.

Cis-directed cleavage and nonstoichiometric abundances of 21-nucleotide reproductive phased small interfering RNAs in grasses.

Post-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNAs that are processed into 21- and 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275. We characterized these 'reproductive phasiRNAs' from rice (Oryza sativa) panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions. We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production. We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived 'top' strand phasiRNAs are consistently higher in abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.