Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific ...genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyADelta plcABCDelta. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PAC:I restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PAC:I site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.
Abstract
RIG-I (retinoic acid inducible gene-I) can sense subtle differences between endogenous and viral RNA in the cytoplasm, triggering an anti-viral immune response through induction of type I ...interferons (IFN) and other inflammatory mediators. Multiple crystal and cryo-EM structures of RIG-I suggested a mechanism in which the C-terminal domain (CTD) is responsible for the recognition of viral RNA with a 5′-triphoshate modification, while the CARD domains serve as a trigger for downstream signaling, leading to the induction of type I IFN. However, to date contradicting conclusions have been reached around the role of ATP in the mechanism of the CARD domains ejection from RIG-I’s autoinhibited state. Here we present an application of NMR spectroscopy to investigate changes induced by the binding of 5′-triphosphate and 5′-OH dsRNA, both in the presence and absence of nucleotides, to full length RIG-I with all its methionine residues selectively labeled (Met-ϵ-13CH3). With this approach we were able to identify residues on the CTD, helicase domain, and CARDs that served as probes to sense RNA-induced conformational changes in those respective regions. Our results were analyzed in the context of either agonistic or antagonistic RNAs, by and large supporting a mechanism proposed by the Pyle Lab in which CARD release is primarily dependent on the RNA binding event.
Graphical Abstract
Graphical Abstract
Persistent genital arousal disorder (PGAD), a condition of unwanted, unremitting sensations of genital arousal, is associated with a significant, negative psychosocial impact that may include ...emotional lability, catastrophization, and suicidal ideation. Despite being first reported in 2001, PGAD remains poorly understood.
To characterize this complex condition more accurately, review the epidemiology and pathophysiology, and provide new nomenclature and guidance for evidence-based management.
A panel of experts reviewed pertinent literature, discussed research and clinical experience, and used a modified Delphi method to reach consensus concerning nomenclature, etiology, and associated factors. Levels of evidence and grades of recommendation were assigned for diagnosis and treatment.
The nomenclature of PGAD was broadened to include genito-pelvic dysesthesia (GPD), and a new biopsychosocial diagnostic and treatment algorithm for PGAD/GPD was developed.
The panel recognized that the term PGAD does not fully characterize the constellation of GPD symptoms experienced by patients. Therefore, the more inclusive term PGAD/GPD was adopted, which maintains the primacy of the distressing arousal symptoms and acknowledges associated bothersome GPD. While there are diverse biopsychosocial contributors, there is a common underlying neurologic basis attributable to spontaneous intense activity of the genito-pelvic region represented in the somatosensory cortex and its projections. A process of care diagnostic and treatment strategy was developed to guide the clinician, whenever possible, by localizing the symptoms as originating in any of five regions: (i) end organ, (ii) pelvis/perineum, (iii) cauda equina, (iv) spinal cord, and (v) brain. Psychological treatment strategies were considered critical and should be performed in conjunction with medical strategies. Pharmaceutical interventions may be used based on their site and mechanism of action to reduce patients’ symptoms and the associated bother and distress.
The process of care for PGAD/GPD uses a personalized, biopsychosocial approach for diagnosis and treatment.
Strengths and Limitations: Strengths include characterization of the condition by consensus, analysis, and recommendation of a new nomenclature and a rational basis for diagnosis and treatment. Future investigations into etiology and treatment outcomes are recommended. The main limitations are the dearth of knowledge concerning this condition and that the current literature consists primarily of case reports and expert opinion.
We provide, for the first time, an expert consensus review of the epidemiology and pathophysiology and the development of a new nomenclature and rational algorithm for management of this extremely distressing sexual health condition that may be more prevalent than previously recognized.
Goldstein I, Komisaruk BR, Pukall CF, et al. International Society for the Study of Women’s Sexual Health (ISSWSH) Review of Epidemiology and Pathophysiology, and a Consensus Nomenclature and Process of Care for the Management of Persistent Genital Arousal Disorder/Genito-Pelvic Dysesthesia (PGAD/GPD). J Sex Med 2021;18:665–697.
Taller adult height is associated with lower risks of ischemic heart disease in mendelian randomization (MR) studies, but little is known about the causal relevance of height for different subtypes ...of ischemic stroke. The present study examined the causal relevance of height for different subtypes of ischemic stroke.
Height-associated genetic variants (up to 2,337) from previous genome-wide association studies (GWASs) were used to construct genetic instruments in different ancestral populations. Two-sample MR approaches were used to examine the associations of genetically determined height with ischemic stroke and its subtypes (cardioembolic stroke, large-artery stroke, and small-vessel stroke) in multiple ancestries (the MEGASTROKE consortium, which included genome-wide studies of stroke and stroke subtypes: 60,341 ischemic stroke cases) supported by additional cases in individuals of white British ancestry (UK Biobank UKB: 4,055 cases) and Chinese ancestry (China Kadoorie Biobank CKB: 10,297 cases). The associations of genetically determined height with established cardiovascular and other risk factors were examined in 336,750 participants from UKB and 58,277 participants from CKB. In MEGASTROKE, genetically determined height was associated with a 4% lower risk (odds ratio OR 0.96; 95% confidence interval CI 0.94, 0.99; p = 0.007) of ischemic stroke per 1 standard deviation (SD) taller height, but this masked a much stronger positive association of height with cardioembolic stroke (13% higher risk, OR 1.13 95% CI 1.07, 1.19, p < 0.001) and stronger inverse associations with large-artery stroke (11% lower risk, OR 0.89 0.84, 0.95, p < 0.001) and small-vessel stroke (13% lower risk, OR 0.87 0.83, 0.92, p < 0.001). The findings in both UKB and CKB were directionally concordant with those observed in MEGASTROKE, but did not reach statistical significance: For presumed cardioembolic stroke, the ORs were 1.08 (95% CI 0.86, 1.35; p = 0.53) in UKB and 1.20 (0.77, 1.85; p = 0.43) in CKB; for other subtypes of ischemic stroke in UKB, the OR was 0.97 (95% CI 0.90, 1.05; p = 0.49); and for other nonlacunar stroke and lacunar stroke in CKB, the ORs were 0.89 (0.80, 1.00; p = 0.06) and 0.99 (0.88, 1.12; p = 0.85), respectively. In addition, genetically determined height was also positively associated with atrial fibrillation (available only in UKB), and with lean body mass and lung function, and inversely associated with low-density lipoprotein (LDL) cholesterol in both British and Chinese ancestries. Limitations of this study include potential bias from assortative mating or pleiotropic effects of genetic variants and incomplete generalizability of genetic instruments to different populations.
The findings provide support for a causal association of taller adult height with higher risk of cardioembolic stroke and lower risk of other ischemic stroke subtypes in diverse ancestries. Further research is needed to understand the shared biological and physical pathways underlying the associations between height and stroke risks, which could identify potential targets for treatments to prevent stroke.
Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by
biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, ...synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of
biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit
biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis.
The glycolytic phenotype is a widespread phenomenon in solid cancer forms, including breast cancer. Dichloroacetate (DCA) has recently been proposed as a novel and relatively non-toxic anti-cancer ...agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. We have examined the effect of DCA against breast cancer cells, including in a highly metastatic in vivo model. The growth of several breast cancer cell lines was found to be inhibited by DCA in vitro. Further examination of 13762 MAT rat mammary adenocarcinoma cells found that reversal of the glycolytic phenotype by DCA correlated with the inhibition of proliferation without any increase in cell death. This was despite a small but significant increase in caspase 3/7 activity, which may sensitize cancer cells to other apoptotic triggers. In vivo, DCA caused a 58% reduction in the number of lung metastases observed macroscopically after injection of 13762 MAT cells into the tail vein of rats (P = 0.0001, n ≥ 9 per group). These results demonstrate that DCA has anti-proliferative properties in addition to pro-apoptotic properties, and can be effective against highly metastatic disease in vivo, highlighting its potential for clinical use.
Heme oxygenase-1 (HMOX1) plays a critical role in the protection of cells, and the inducible enzyme is implicated in a spectrum of human diseases. The increasing prevalence of cardiovascular and ...metabolic morbidities, for which current treatment approaches are not optimal, emphasizes the necessity to better understand key players such as HMOX1 that may be therapeutic targets.
HMOX1 is a dynamic protein that can undergo post-translational and structural modifications which modulate HMOX1 function. Moreover, trafficking from the endoplasmic reticulum to other cellular compartments, including the nucleus, highlights that HMOX1 may play roles other than the catabolism of heme.
The ability of HMOX1 to be induced by a variety of stressors, in an equally wide variety of tissues and cell types, represents an obstacle for the therapeutic exploitation of the enzyme. Any capacity to modulate HMOX1 in cardiovascular and metabolic diseases should be tempered with an appreciation that HMOX1 may have an impact on cancer. Moreover, the potential for heme catabolism end products, such as carbon monoxide, to amplify the HMOX1 stress response should be considered.
A more complete understanding of HMOX1 modifications and the properties that they impart is necessary. Delineating these parameters will provide a clearer picture of the opportunities to modulate HMOX1 in human disease.
This study aimed to determine the prevalence of treated and untreated substance use disorders among Medicare beneficiaries, the characteristics of Medicare beneficiaries with substance use disorders, ...and reasons for their unmet needs.
This study used data from the National Survey of Drug Use and Health, 2015–2019. Substance use disorder was defined based on DSM-IV dependence or abuse criteria. Descriptive analyses were conducted in 2021, including testing for differences in unadjusted means.
Approximately 1.7 million Medicare beneficiaries were estimated to have past-year substance use disorder (8% of Medicare beneficiaries aged <65 years and 2% aged ≥65 years). Overall, 77% had an alcohol use condition, 16% had a prescription drug use condition, and 10% had a marijuana use condition. Of those who had past-year substance use disorder, 11% received treatment for their condition. Common reasons for not receiving treatment were lack of motivation (41%), financial barriers (33%), concern about what others might think (24%), logistical barriers such as lack of transportation (21%), and uncertainty about treatment efficacy (13%). Medicare beneficiaries with substance use disorders were more than twice as likely to have past-year serious psychological distress as those without substance use disorders (44% vs 21%, p<0.001 for those aged <65 years; 14% vs 4%, p<0.001 for those aged ≥65 years). Percentages of past-year suicidal ideation were also much higher among Medicare beneficiaries with substance use disorders than without (24% vs 6%, p<0.001 for those aged <65 years; 7% vs 2%, p=0.006 for those aged ≥65 years).
Few Medicare beneficiaries who need substance use disorder treatment receive it. Reducing Medicare coverage gaps and stigma may help meet this need.
Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed ...outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (
), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The
gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.
Honey bees have suffered dramatic losses in recent years, largely due to multiple stressors underpinned by poor nutrition 1. Nutritional stress especially harms larvae, who mature into workers unable ...to meet the needs of their colony 2. In this study, we characterize the metabolic capabilities of a honey bee larvae-associated bacterium, Bombella apis (formerly Parasaccharibacter apium), and its effects on the nutritional resilience of larvae. We found that B. apis is the only bacterium associated with larvae that can withstand the antimicrobial larval diet. Further, we found that B. apis can synthesize all essential amino acids and significantly alters the amino acid content of synthetic larval diet, largely by supplying the essential amino acid lysine. Analyses of gene gain/loss across the phylogeny suggest that four amino acid transporters were gained in recent B. apis ancestors. In addition, the transporter LysE is conserved across all sequenced strains of B. apis. Finally, we tested the impact of B. apis on developing honey bee larvae subjected to nutritional stress and found that larvae supplemented with B. apis are bolstered against mass reduction despite limited nutrition. Together, these data suggest a novel role of B. apis as a nutritional mutualist of honey bee larvae.
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