Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

Systematic review of non-invasive ventilation and craniofacial fractures: a multidisciplinary perspective and recommendations for management.

The anatomically complex craniofacial skeleton demands special consideration when caring for cases of polytrauma or medically compromised patients with craniofacial fractures. This paper utilises a systematic review and multidisciplinary opinions to create an algorithm for the hospital-based care of patients with craniofacial fractures (base of skull, orbit, paranasal sinus, and mandible) who require non-invasive ventilation (NIV). Each fracture location has a unique predisposition to a different type of emphysema and associated morbidity. The risk of developing emphysema, combined with its potential severity, is stratified against the harm of not providing NIV for the holistic care of the patient. The aim of this paper is to synthesise evidence from a systematic review of existing literature with multidisciplinary opinions to develop a concise algorithm that outlines the optimal treatment of patients with craniofacial fractures who require NIV.

In vitro activity of omadacycline against clinical isolates of Nocardia.

Nocardiosis typically requires a prolonged treatment duration of ≥6 months and initial combination therapy with 2-3 antibiotics. First-line regimens for nocardiosis are associated with considerable toxicity; therefore, alternative therapies are needed. Omadacycline is an aminomethylcycline with broad antimicrobial activity whose in vitro activity against Nocardia species has not been formally assessed. The in vitro potency of omadacycline was evaluated against 300 Nocardia clinical isolates by broth microdilution. The most common Nocardia species tested were N. cyriacigeorgica (21%), N. nova (20%), and N. farcinica (12%). The most common specimens were respiratory (178 isolates, 59%) and wound (57 isolates, 19%). Omadacycline minimum inhibitory concentrations (MICs) across all Nocardia species ranged from 0.06 µg/mL to 8 µg/mL, with an MIC50 of 2 µg/mL and MIC90 of 4 µg/mL. The lowest MICs were found among N. paucivorans (MIC50 = 0.25 µg/mL, MIC90 = 0.25 µg/mL), N. asiatica (MIC50 = 0.25 µg/mL, MIC90 = 1 µg/mL), N. abscessus complex (MIC50 = 0.5 µg/mL, MIC90 = 1 µg/mL), N. beijingensis (MIC50 = 0.5 µg/mL, MIC90 = 2 µg/mL), and N. otitidiscaviarum (MIC50 = 1 µg/mL, MIC90 = 2 µg/mL). The highest MICs were found among N. farcinica (MIC50 = 4 µg/mL, MIC90 = 8 µg/mL). In vitro potency differed by species among Nocardia clinical isolates. Further studies are warranted to evaluate the potential clinical utility of omadacycline for nocardiosis.

New Directions in Infection-Associated Ischemic Stroke.

The relationship between infections and stroke has not been fully characterized, probably delaying the development of specific treatments. This narrative review addresses mechanisms of stroke linked to infections, including hypercoagulability, endothelial dysfunction, vasculitis, and impaired thrombolysis. SARS-CoV-2, the virus that causes COVID-19, may promote the development of stroke, which may represent its most severe neurological complication. The development of specific therapies for infection-associated stroke remains a profound challenge. Perhaps the most important remaining issue is the distinction between infections that trigger a stroke versus infections that are truly incidental. This distinction likely requires the establishment of appropriate biomarkers, candidates of which are elevated levels of fibrin D-dimer and anticardiolipin/antiphospholipid antibodies. These candidate biomarkers might have potential use in identifying pathogenic infections preceding stroke, which is a precursor to establishing specific therapies for this syndrome.

Comparative analysis of a rapid diagnostic test and scoring tools for ESBL detection in Enterobacterales bloodstream infections for optimizing antimicrobial therapy.

IMPORTANCE: Our study addresses a significant issue in the medical and scientific community-the delayed administration of appropriate antimicrobial treatments due to the time-consuming process of phenotypic susceptibility data collection in gram-negative bloodstream infections. Our research indicates that a multiplex PCR rapid diagnostic test (RDT) significantly outperformed two clinical scoring tools in predicting ceftriaxone susceptibility. Multiplex PCR also led to reduced instances of undertreatment with ceftriaxone and minimized overtreatment with carbapenems. Furthermore, multiplex PCR demonstrated high sensitivity and specificity in predicting ceftriaxone susceptibility. The results of our study underscore the potential RDTs to reduce the time to appropriate antimicrobial therapy, leading to improved patient outcomes and reduced healthcare costs.

Dynamics and recognition of homeodomain containing protein-DNA complex of IRX4.

Iroquois Homeobox 4 (IRX4) belongs to a family of homeobox TFs having roles in embryogenesis, cell specification, and organ development. Recently, large scale genome-wide association studies and epigenetic studies have highlighted the role of IRX4 and its associated variants in prostate cancer. No studies have investigated and characterized the structural aspect of the IRX4 homeodomain and its potential to bind to DNA. The current study uses sequence analysis, homology modeling, and molecular dynamics simulations to explore IRX4 homeodomain-DNA recognition mechanisms and the role of somatic mutations affecting these interactions. Using publicly available databases, gene expression of IRX4 was found in different tissues, including prostate, heart, skin, vagina, and the protein expression was found in cancer cell lines (HCT166, HEK293), B cells, ascitic fluid, and brain. Sequence conservation of the homeodomain shed light on the importance of N- and C-terminal residues involved in DNA binding. The specificity of IRX4 homodimer bound to consensus human DNA sequence was confirmed by molecular dynamics simulations, representing the role of conserved amino acids including R145, A194, N195, S190, R198, and R199 in binding to DNA. Additional N-terminal residues like T144 and G143 were also found to have specific interactions highlighting the importance of N-terminus of the homeodomain in DNA recognition. Additionally, the effects of somatic mutations, including the conserved Arginine (R145, R198, and R199) residues on DNA binding elucidated the importance of these residues in stabilizing the protein-DNA complex. Secondary structure and hydrogen bonding analysis showed the roles of specific residues (R145, T191, A194, N195, R198, and R199) in maintaining the homogeneity of the structure and its interaction with DNA. The differences in relative binding free energies of all the mutants shed light on the structural modularity of this protein and the dynamics behind protein-DNA interaction. We also have predicted that the C-terminal sequence of the IRX4 homeodomain could act as a potential cell-penetrating peptide, emphasizing the role these small peptides could play in targeting homeobox TFs.

Erythrocyte-brain endothelial interactions induce microglial responses and cerebral microhemorrhages in vivo.

BACKGROUND: Cerebral microhemorrhages (CMH) are associated with stroke, cognitive decline, and normal aging. Our previous study shows that the interaction between oxidatively stressed red blood cells (RBC) and cerebral endothelium may underlie CMH development. However, the real-time examination of altered RBC-brain endothelial interactions in vivo, and their relationship with clearance of stalled RBC, microglial responses, and CMH development, has not been reported. METHODS: RBC were oxidatively stressed using tert-butylhydroperoxide (t-BHP), fluorescently labeled and injected into adult Tie2-GFP mice. In vivo two-photon imaging and ex vivo confocal microscopy were used to evaluate the temporal profile of RBC-brain endothelial interactions associated with oxidatively stressed RBC. Their relationship with microglial activation and CMH was examined with post-mortem histology. RESULTS: Oxidatively stressed RBC stall significantly and rapidly in cerebral vessels in mice, accompanied by decreased blood flow velocity which recovers at 5 days. Post-mortem histology confirms significantly greater RBC-cerebral endothelial interactions and microglial activation at 24 h after t-BHP-treated RBC injection, which persist at 7 days. Furthermore, significant CMH develop in the absence of blood-brain barrier leakage after t-BHP-RBC injection. CONCLUSIONS: Our in vivo and ex vivo findings show the stalling and clearance of oxidatively stressed RBC in cerebral capillaries, highlighting the significance of microglial responses and altered RBC-brain endothelial interactions in CMH development. Our study provides novel mechanistic insight into CMH associated with pathological conditions with increased RBC-brain endothelial interactions.

Determinants of lipid clinic referral and attendance in a multi-ethnic adult population in south London: a cross-sectional study.

BACKGROUND: Primary dyslipidaemias, including familial hypercholesterolaemia, are underdiagnosed genetic disorders that substantially increase risk for premature coronary artery disease in adults. Early identification of primary dyslipidaemias via lipid clinic referral optimises patient management and enables cascade screening of relatives. Improving the identification of primary dyslipidaemias, and understanding disparities in ascertainment and management, is an NHS priority. We aimed to assess determinants of lipid clinic referral or attendance (LCR) in ethnically diverse adults. METHODS: We did a retrospective cross-sectional study using the Lambeth DataNet containing anonymised data from 41 general practitioner (GP) practices in south London. We looked at referral data for adult patients aged 18 years and older from Jan 1, 1995, until May 14, 2018. LCR was the main outcome. We used sequential multilevel logistic regression models adjusted for practice effects to estimate the odds of LCR assessed across six ethnic groups (reference group White) and patient-level factors (demographic, socioeconomic, lifestyle, comorbidities, total cholesterol [TC] >7·5mmol/L, statin prescription, and practice factors). The study was approved by NHS South East London Clinical Commissioning Group (CCG) and NHS Lambeth CCG. FINDINGS: 780 (0·23%) of 332 357 adult patients were coded as referred (n=538) or seen (n=252) in a lipid clinic. 164 487 (46·49%) were women (appendix). The fully adjusted model for odds of LCR showed the following significant associations for age (odds ratio [OR] 0·96, 95% CI 0·96-0·97, p<0·001); Black, African, Caribbean, or Black-British ethnicity (0·67, 0·53-0·84, p=0·001); ex-smoker status (1·29, 1·05-1·57, p=0·014); TC higher than 7·5 mmol/L (12·18, 9·60-15·45, p<0·001); statin prescription (14·01, 10·85-18·10, p<0·001); diabetes (0·72, 0·58-0·91, p=0·005); high-frequency GP attendance at seven or more GP consultations in the past year (1·49, 1·21-1·84, p<0·001); high GP-density (0·5-0·99 full-time equivalent GPs per 1000 patients; 2·70, 1·23-5·92, p=0·013). Sensitivity analyses for LCR restricted to familial hypercholesterolaemia-coded patients (n=581) found associations with TC higher than 7·5 mmol/L (4·26, 1·89-9·62, p<0·001), statin prescription (16·96, 2·19-131·36, p=0·007), and high GP-density (5·73, 1·27-25·93, p=0·023), with no significant associations with ethnicity. The relative contribution of GP practices to LCR was 6·32% of the total variance. There were no significant interactions between ethnicity and deprivation, age, or obesity. INTERPRETATION: While interpretation is limited by the accuracy and completeness of coded records, the study showed factors associated with a higher likelihood of LCR included individuals recorded as having TC higher than 7·5 mmol/L, statin prescription, ex-smoker status, high-frequency GP attendance, and registration at a GP practice with 0·5-0·99 GP density. Patients with increasing age; Black, African, Caribbean, or Black-British ethnicity patients; and patients with diabetes had lower odds of LCR. Finally, the difference in odds of LCR between Black and White patients highlights potential health inequalities. FUNDING: NHS Race & Health Observatory.

Targeting the COMMD4-H2B protein complex in lung cancer.

BACKGROUND: Lung cancer is the biggest cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85-90% of all lung cancers. Identification of novel therapeutic targets are required as drug resistance impairs chemotherapy effectiveness. COMMD4 is a potential NSCLC therapeutic target. The aims of this study were to investigate the COMMD4-H2B binding pose and develop a short H2B peptide that disrupts the COMMD4-H2B interaction and mimics COMMD4 siRNA depletion. METHODS: Molecular modelling, in vitro binding and site-directed mutagenesis were used to identify the COMMD4-H2B binding pose and develop a H2B peptide to inhibit the COMMD4-H2B interaction. Cell viability, DNA repair and mitotic catastrophe assays were performed to determine whether this peptide can specially kill NSCLC cells. RESULTS: Based on the COMMD4-H2B binding pose, we have identified a H2B peptide that inhibits COMMD4-H2B by directly binding to COMMD4 on its H2B binding binding site, both in vitro and in vivo. Treatment of NSCLC cell lines with this peptide resulted in increased sensitivity to ionising radiation, increased DNA double-strand breaks and induction of mitotic catastrophe in NSCLC cell lines. CONCLUSIONS: Our data shows that COMMD4-H2B represents a novel potential NSCLC therapeutic target.

The potential impact of periodontitis on cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is a term used to describe abnormalities in the intracranial microvasculature affecting small arteries, arterioles, capillaries, and venules. The etiology of these conditions is not fully understood but inflammation appears to play a significant role. Periodontal diseases have been associated with conditions such as stroke and dementia, which are clinical consequences of CSVD. Periodontitis is a highly prevalent chronic multifactorial inflammatory disease regulated by the host immune response against pathogenic bacterial colonization around the teeth. The inflammatory response and the microbial dysbiosis produce pro-inflammatory cytokines that can reach the brain and promote local changes. This review will explore the potential association between periodontitis and CSVD by assessing the impact of periodontitis-induced inflammation and periodontopathogenic bacteria on the underlying mechanisms leading to CSVD. Given the association of periodontitis with stroke and dementia, which are clinical features of CSVD, it may be possible to suggest a link with CSVD. Current evidence linking periodontitis with neuroimaging findings of CSVD enforces the possible link between these conditions.

Changes in beat-to-beat blood pressure and pulse rate variability following stroke.

Associations between cerebrovascular disease and impaired autonomic function and cerebrovascular reactivity have led to increased interest in variability of heart rate (HRV) and blood pressure (BPV) following stroke. In this study, beat-to-beat pulse rate variability (PRV) and BPV were measured in clinically stable stroke patients (6 ischemic, 2 hemorrhagic) at least one year after their last cerebrovascular event. Beat-to-beat blood pressure (BP) measurements were collected from subjects while resting in the sitting position for one hour. Compared with healthy controls, stroke patients exhibited significantly greater time-domain (standard deviation, coefficient of variation, average real variability) and normalized high-frequency BPV (all p < 0.05). Stroke patients also exhibited lower LF:HF ratios than control subjects (p = 0.003). No significant differences were observed in PRV between the two groups, suggesting that BPV may be a more sensitive biomarker of cerebrovascular function in long-term post-stroke patients. Given a paucity of existing literature investigating beat-to-beat BPV in clinically stable post-stroke patients long (> 1 year) after their cerebrovascular events, this pilot study can help inform future studies investigating the mechanisms and effects of BPV in stroke. Elucidating this physiology may facilitate long-term patient monitoring and pharmacological management to mitigate the risk for recurrent stroke.

Genomic Characterization of 2 Cutibacterium acnes Isolates from a Surgical Site Infection Reveals Large Genomic Inversion.

Background: Cutibacterium acnes is a common commensal of human skin but may also present as an opportunistic pathogen in prosthetic joint and wound infections. Unfortunately, few complete genomes of C. acnes are publicly available, and even fewer are of isolates associated with infection. Here we report the isolation, characterization, and complete genomes of 2 C. acnes isolates from a surgical site infection of an elbow. Methods: We used standard microbiological methods for phenotypic characterization and performed whole genome sequencing on 2 C. acnes isolates using a combination of short-read and long-read sequencing. Results: Antibiotic susceptibility testing showed beta-lactamase negative and low minimal inhibitory concentrations to all antibiotics tested, with the exception of metronidazole. We assembled complete genomes of the 2 isolates, which are approximately 2.5 megabases in length. The isolates belong to the single-locus sequence type (SLST) H1 and the multi-locus sequence type (MLST) IB. Both isolates have similar composition of known virulence genes, and we found no evidence of plasmids but did find phage-associated genes. Notably, the 2 genomes are 99.97% identical but contain a large genomic inversion encompassing approximately half of the genome. Conclusions: This is the first characterization of this large-scale genomic inversion in nearly identical isolates from the same wound. This report adds to the limited numbers of publicly available infection-associated complete genomes of C. acnes.

Resistance mechanisms for Gram-negative bacteria-specific lipopeptides, turnercyclamycins, differ from that of colistin.

IMPORTANCE: Bacterial resistance to antibiotics is a crisis. Acinetobacter baumannii is among the CDC urgent threat pathogens in part for this reason. Lipopeptides known as turnercyclamycins are produced by symbiotic bacteria that normally live in marine mollusks, where they may be involved in shaping their symbiotic niche. Turnercyclamycins killed Gram-negative pathogens including drug-resistant Acinetobacter, but how do the mechanisms of resistance compare to other lipopeptide drugs? Here, we define resistance from a truncation of MlaA, a protein involved in regulating bacterial membrane phospholipids. Intriguingly, this resistance mechanism only affected one turnercyclamycin variant, which differed only in two atoms in the lipid tail of the compounds. We could not obtain significant resistance to the second turnercyclamycin variant, which was also effective in an infection model. This study reveals an unexpected subtlety in resistance to lipopeptide antibiotics, which may be useful in the design and development of antibiotics to combat drug resistance.

Enterobacterial common antigen biosynthesis in Yersinia pestis is tied to antimicrobial peptide resistance.

Resistance to antimicrobial peptides (AMPs) plays an important role in allowing Yersinia pestis to maintain a successful infection in the flea vector Xenopsylla cheopis . Mutants that are unable to modify lipid A in their outer membrane with aminoarabinose (Ara4N), showed increased sensitivity to AMPs such as polymyxin B (PB), as well as decreased survival in fleas. A deletion mutant of wecE , a gene involved in biosynthesis of enterobacterial common antigen (ECA), also displayed hypersusceptibility to PB in vitro. Additional mutants in the ECA biosynthetic pathway were generated, some designed to cause accumulation of intermediate products that sequester undecaprenyl phosphate (Und-P), a lipid carrier that is also used in numerous other pathways, including for peptidoglycan, O-antigen, and Ara4N biosynthesis. Mutants that accumulate Und-PP-linked intermediates (ECA-lipid II) showed increased susceptibility to PB, reduced Ara4N-modified lipid A, altered cell morphology, and decreased ability to maintain flea infections. These effects are consistent with a model where Y. pestis has a sufficiently limited free Und-P pool such that sequestration of Und-P as ECA-lipid II prevents adequate Ara4N biosynthesis, ultimately resulting in AMP hypersusceptibility.

Evaluating the sensitivity and specificity of dried blood spots for serological testing of HIV, syphilis, hepatitis B and C Elecsys assays on the Roche Cobas system.

This study was performed to validate a dried blood spot (DBS) method for the serological screening of HIV, syphilis, hepatitis B and C. It included 250 paired DBS and serum samples and 116 unpaired DBS samples from 366 unique patients from two laboratories between 8 October and 2 November 2021. As determined by original test request, these were tested using a DBS method for HIV Ag/Ab (n=216), anti-treponemal Ab (n=166), hepatitis B sAg (n=100), and hepatitis C Ab (n=100) Elecsys assays on the Roche Cobas automated platform. Using the manufacturer's (serum) cut-off for reactivity ('positivity'), the sensitivity and specificity of DBS testing compared with serum were: for HIV Ag/Ab 100% and 100%, for anti-treponemal Ab 68.3% and 100%, for hepatitis B sAg 95.9% and 100%, and for hepatitis C Ab 84.0% and 100%, respectively. Adjusting the assay cut-off using receiver operator curve analysis increased sensitivity of DBS testing for anti-treponemal Ab to 90.0%, hepatitis B sAg to 97.9% and hepatitis C Ab to 94.0% whilst maintaining specificity of 98.8%, 100% and 100%, respectively. With optimisation of assay cut-off, DBS can perform comparably with serum for serological testing for HIV, syphilis, hepatitis B and C and may be a valuable tool in increasing access to testing in New Zealand.

The 2022 FASEB Virtual Catalyst Conference on the Cardiac Interatrial Septum and Stroke Risk, December 7, 2022.

There is emerging evidence that the cardiac interatrial septum has an important role as a thromboembolic source for ischemic strokes. There is little consensus on treatment of patients with different cardiac interatrial morphologies or pathologies who have had stroke. In this paper, we summarize the important background, diagnostic, and treatment considerations for this patient population as presented during the Federation of American Societies for Experimental Biology (FASEB) Virtual Catalytic Conference on the Cardiac Interatrial Septum and Stroke Risk, held on December 7, 2022. During this conference, many aspects of the cardiac interatrial septum were discussed. Among these were the embryogenesis of the interatrial septum and development of anatomic variants such as patent foramen ovale and left atrial septal pouch. Also addressed were various mechanisms of injury such as shunting physiologies and the consequences that can result from anatomic variants, as well as imaging considerations in echocardiography, computed tomography, and magnetic resonance imaging. Treatment options including anticoagulation and closure were addressed, as well as an in-depth discussion on whether the left atrial septal pouch is a stroke risk factor. These issues were discussed and debated by multiple experts from neurology, cardiology, and radiology.

Saddle Thigh Block Design Can Influence Rider and Horse Biomechanics.

The association between rider-saddle interaction and horse kinematics has been little studied. It was hypothesized that differences in a thigh block design would influence (a) rider-saddle interface pressures, (b) rider kinematics, and (c) equine limb/spinal kinematics. Eighteen elite sport horses/riders were trotted using correctly fitted dressage saddles with thigh blocks S (vertical face) and F (deformable face). Contact area, mean, and peak pressure between rider and saddle were determined using an on-saddle pressure mat. Spherical markers allowed for the measurement of horse/rider kinematics using two-dimensional video analysis. The kinematics of the equine thoracolumbosacral spine were obtained using skin-mounted inertial measuring units. Results were compared between thigh blocks (paired t-test p ≤ 0.05). With F, the contact area, mean, and peak pressure between rider and saddle were significantly higher (p = 0.0001), and the rider trunk anterior tilt was reduced, indicating altered rider-saddle interaction. The horse thoracic axial rotation and flexion/extension were reduced (p = 0.01-0.03), caudal thoracic and lumbar lateral bend was increased (p = 0.02-0.04), and carpal flexion increased (p = 0.01-0.05) with F compared to S. During straight-line locomotion when in sitting trot, thigh block F was associated with altered rider-saddle interaction and rider and equine kinematics, leading to a more consistent rider-saddle interface, a more upright rider trunk during stance, an increased horse thoracic stability and lumbar lateral bend, and forelimb flexion, supporting the importance of optimising rider-saddle-horse interaction.

Elevated Intraocular Pressure in Periorbital Sweet's Syndrome.

A 66-year-old immunocompromised man presented with cellulitis around the left eye that was initially concerning for necrotizing fasciitis. Exam findings were remarkable for exquisite periocular tenderness with rigid, immobile eyelids resulting from severe erythema, edema, and induration. Given the concern for orbital compartment syndrome and a necrotizing infection, the patient was taken urgently to the operating room for debridement of the eyelid skin as well as an urgent lateral canthotomy and cantholysis. His eye exam revealed 360° of hemorrhagic chemosis, no relative afferent pupillary defect, and an ipsilateral elevated intraocular pressure of 35 mm Hg. No visual acuity measurement could be obtained secondary to the patient's altered mental status. His intraocular pressure normalized after treatment with antihypertensive drops and further extension of the canthotomy. Histopathological analysis showed extensive neutrophilic infiltrate of the dermis which was compatible with a diagnosis of Sweet's syndrome.