SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Is there 'trustworthy' evidence for using manual therapy to treat patients with shoulder dysfunction?: A systematic review.

The primary objective of this review was to create a 'trustworthy,' living systematic review and meta-analysis for the application of manual therapy interventions in treating patients with shoulder dysfunction. Included studies were English-language randomized controlled trials published between 1/1/2010 and 8/3/2023, with searches performed in: PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), CINHAL, ProQuest Nursing & Allied Health, EBSCO Medline, and PEDro. The population of focus included adults 18 years and older with musculoskeletal impairments related to shoulder dysfunction. Our primary outcomes included pain and region-specific outcome measures. We excluded trials, including participants having shoulder dysfunction resulting from surgery, radicular pain, instability/dislocation, fracture, lymphedema, and radiation. Our screening methodology was based upon a previously published 'trustworthy' systematic review protocol. This included the application of our PICOTS criteria in addition to screening for prospective clinical trial registration and following of prospective intent, as well as assessment of PEDro scores, risk-of-bias ratings, GRADE scoring, and examination of confidence in estimated effects. Twenty-six randomized controlled trials met our PICOTS criteria; however, only 15 of these were registered. Only three were registered prospectively. Two of these did not have discussions and conclusions that aligned with their primary outcome. The remaining single study was found to have a high risk-of-bias, meaning the remainder of the protocol could not be employed and that no randomized controlled trials could undergo further assessment or meta-analysis. The results of this systematic review indicate there are no 'trustworthy' randomized controlled trials examining the effectiveness of manual therapy interventions for the treatment of patients with shoulder dysfunction, as defined by the prospectively established methodology. Therefore, these findings signal that creating a 'trustworthy,' living systematic review on this clinically relevant topic is not yet possible due to a lack of 'trustworthy' randomized controlled trials.

Does manual therapy meaningfully change quantitative sensory testing and patient reported outcome measures in patients with musculoskeletal impairments related to the spine?: A 'trustworthy' systematic review and meta-analysis.

OBJECTIVES: To perform a 'trustworthy' systematic review (SR) with meta-analysis on the potential mechanisms of manual therapy used to treat spinal impairments. DESIGN: SR with meta-analysis. LITERATURE SEARCH: Articles published between January 2010 and October 2022 from CENTRAL, CINAHL, MEDLINE, PubMed, ProQuest, and PEDro. METHODS: This SR included English-language randomized clinical trials (RCTs) involving manual therapy to treat spinal impairments in adults. The primary outcome was pressure pain thresholds (PPTs). To synthesize RCTs with high confidence in estimated effects using the GRADE, RCTs with questionable prospective, external, and internal validity, and high risk of bias (RoB) were excluded. RESULTS: Following title and abstract screening, 89 full-text RCTs were reviewed. Twenty-two studies included the criteria of interest. Sixteen were not prospectively registered, two contained discussion/conclusions judged to be inconsistent with the registry, and one was rated as having a high RoB. Three studies met the inclusion criteria; heterogeneous interventions and locations for PPT testing prevented synthesis into practice recommendations. The two studies with high confidence in estimated effects had small effect sizes, and one study had confidence intervals that crossed zero for the outcome measures of interest. DISCUSSION: Standardized PPT testing, as a potential measure of centrally mediated pain, could provide clues regarding the mechanisms of manual therapy or help identify/refine research questions. CONCLUSION: High-quality RCTs could not be synthesized into strong conclusions secondary to the dissimilarity in research designs. Future research regarding quantitative sensory testing should develop RCTs with high confidence in estimated effects that can be translated into strong recommendations.

Anaphylatoxin signaling activates macrophages to control intracellular Rickettsia proliferation.

IMPORTANCE: Pathogenic Rickettsia species are extremely dangerous bacteria that grow within the cytoplasm of host mammalian cells. In most cases, these bacteria are able to overpower the host cell and grow within the protected environment of the cytoplasm. However, a dramatic conflict occurs when Rickettsia encounter innate immune cells; the bacteria can "win" by taking over the host, or the bacteria can "lose" if the host cell efficiently fights the infection. This manuscript examines how the immune complement system is able to detect the presence of Rickettsia and alert nearby cells. Byproducts of complement activation called anaphylatoxins are signals that "activate" innate immune cells to mount an aggressive defensive strategy. This study enhances our collective understanding of the innate immune reaction to intracellular bacteria and will contribute to future efforts at controlling these dangerous infections.

Screening for Latent Tuberculosis Infection in Adults: Updated Evidence Report and Systematic Review for the US Preventive Services Task Force.

Importance: Latent tuberculosis infection (LTBI) can progress to active tuberculosis disease, causing morbidity and mortality. Objective: To review the evidence on benefits and harms of screening for and treatment of LTBI in adults to inform the US Preventive Services Task Force (USPSTF). Data Sources: PubMed/MEDLINE, Cochrane Library, and trial registries through December 3, 2021; references; experts; literature surveillance through January 20, 2023. Study Selection: English-language studies of LTBI screening, LTBI treatment, or accuracy of the tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). Studies of LTBI screening and treatment for public health surveillance or disease management were excluded. Data Extraction and Synthesis: Dual review of abstracts, full-text articles, and study quality; qualitative synthesis of findings; meta-analyses conducted when a sufficient number of similar studies were available. Main Outcomes and Measures: Screening test accuracy; development of active tuberculosis disease, transmission, quality of life, mortality, and harms. Results: A total of 113 publications were included (112 studies; N = 69 009). No studies directly evaluated the benefits and harms of screening. Pooled estimates for sensitivity of the TST were 0.80 (95% CI, 0.74-0.87) at the 5-mm induration threshold, 0.81 (95% CI, 0.76-0.87) at the 10-mm threshold, and 0.60 (95% CI, 0.46-0.74) at the 15-mm threshold. Pooled estimates for sensitivity of IGRA tests ranged from 0.81 (95% CI, 0.79-0.84) to 0.90 (95% CI, 0.87-0.92). Pooled estimates for specificity of screening tests ranged from 0.95 to 0.99. For treatment of LTBI, a large (n = 27 830), good-quality randomized clinical trial found a relative risk (RR) for progression to active tuberculosis at 5 years of 0.35 (95% CI, 0.24-0.52) for 24 weeks of isoniazid compared with placebo (number needed to treat, 112) and an increase in hepatotoxicity (RR, 4.59 [95% CI, 2.03-10.39]; number needed to harm, 279). A previously published meta-analysis reported that multiple regimens were efficacious compared with placebo or no treatment. Meta-analysis found greater risk for hepatotoxicity with isoniazid than with rifampin (pooled RR, 4.22 [95% CI, 2.21-8.06]; n = 7339). Conclusions and Relevance: No studies directly evaluated the benefits and harms of screening for LTBI compared with no screening. TST and IGRAs were moderately sensitive and highly specific. Treatment of LTBI with recommended regimens reduced the risk of progression to active tuberculosis. Isoniazid was associated with higher rates of hepatotoxicity than placebo or rifampin.

Screening for Prediabetes and Type 2 Diabetes in Children and Adolescents: Evidence Report and Systematic Review for the US Preventive Services Task Force.

Importance: Of youths diagnosed with type 2 diabetes, many develop microvascular complications by young adulthood. Objective: To review the evidence on benefits and harms of screening children and adolescents for prediabetes and type 2 diabetes to inform the US Preventive Services Task Force (USPSTF). Data Sources: PubMed/MEDLINE, Cochrane Library, and trial registries through May 3, 2021; references; experts; literature surveillance through July 22, 2022. Study Selection: English-language controlled studies evaluating screening or interventions for prediabetes or type 2 diabetes that was screen detected or recently diagnosed. Data Extraction and Synthesis: Dual review of abstracts, full-text articles, and study quality; qualitative synthesis of findings. Main Outcomes and Measures: Mortality, cardiovascular morbidity, diabetes-related morbidity, development of diabetes, quality of life, and harms. Results: This review included 8 publications (856 participants; mean age, 14 years [range, 10-17 years]). Of those, 6 were from the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study. No eligible studies directly evaluated the benefits or harms of screening. One randomized clinical trial (RCT) (TODAY; n = 699 adolescents with obesity; mean age, 14 years) comparing metformin, metformin plus rosiglitazone, and metformin plus lifestyle intervention reported that 2 youths with recently diagnosed diabetes developed kidney impairment (0 vs 1 vs 1, respectively; P > .99) and 11 developed diabetic ketoacidosis (5 vs 3 vs 3, respectively; P = .70). One RCT of 75 adolescents (mean age, 13 years) with obesity with prediabetes compared an intensive lifestyle intervention with standard care and reported that no participants in either group developed diabetes, although follow-up was only 6 months. Regarding harms of interventions, 2 RCTs assessing different comparisons enrolled youths with recently diagnosed diabetes. Major hypoglycemic events were reported by less than 1% of participants. Minor hypoglycemic events were more common among youths treated with metformin plus rosiglitazone than among those treated with metformin or metformin plus lifestyle intervention in TODAY (8.2% vs 4.3% vs 3.4%, P = .05). In 1 study, gastrointestinal adverse events were more commonly reported by those taking metformin than by those taking placebo (abdominal pain: 25% vs 12%; nausea/vomiting: 17% vs 10%; P not reported). Conclusions and Relevance: No eligible studies directly evaluated the benefits or harms of screening for prediabetes and type 2 diabetes in children and adolescents. For youths with prediabetes or recently diagnosed (not screen-detected) diabetes, the only eligible trials reported few health outcomes and found no difference between groups, although evidence was limited by substantial imprecision and a duration of follow-up likely insufficient to assess health outcomes.

Characterizing the relationship between lesion-activation distance using fMRI and verbal measures in brain tumor patients.

Functional resonance magnetic imaging (fMRI) allows for identification of eloquent cortex in pre-treatment planning. Previous studies have shown a correlation among lesion to activation distance (LAD) measures and morbidity and mortality. This study investigates the relationship between LAD, well-established language centers (Wernicke's and Broca's), and language performance measures. We included a sample population of brain tumor patients that received language fMRI (verbal fluency and sentence verification) for pre-treatment assessment (n = 51). LAD to the nearest language area was measured and divided into groups ≤ 10 mm and > 10 mm. Verbal fluency scores were compared between these groups. Additionally, patients were divided into similar groups based on LAD to either Broca's or Wernicke's areas, and the verbal fluency scores and sentence verification accuracy (n = 29) were subsequently compared between groups. Brain tumor patients with LAD ≤ 10 mm to either language area had significantly lower verbal fluency scores (p = 0.028). The difference in verbal fluency scores between groups with LAD ≤ 10 mm and > 10 mm to Wernicke's area trends toward significance (p = 0.067). The sentence verification accuracy was significantly lower in patients with LAD ≤ 10 mm to either language area (p = 0.039). These findings suggest that there exists a significant relationship between LAD to language centers and measures; greater language deficits are seen when LAD ≤ 10 mm.

The STUN (STop UNhealthy) Alcohol Use Now trial: study protocol for an adaptive randomized trial on dissemination and implementation of screening and management of unhealthy alcohol use in primary care.

BACKGROUND: Unhealthy alcohol use is a leading cause of preventable deaths in the USA and is associated with many societal and health problems. Less than a third of people who visit primary care providers in the USA are asked about or ever discuss alcohol use with a health professional. METHODS/DESIGN: This study is an adaptive, randomized, controlled trial to evaluate the effect of primary care practice facilitation and telehealth services on evidence-based screening, counseling, and pharmacotherapy for unhealthy alcohol use in small-to-medium-sized primary care practices. Study participants will include primary care practices in North Carolina with 10 or fewer providers. All enrolled practices will receive a practice facilitation intervention that includes quality improvement (QI) coaching, electronic health record (EHR) support, training, and expert consultation. After 6 months, practices in the lower 50th percentile (based on performance) will be randomized to continued practice facilitation or provision of telehealth services plus ongoing facilitation for the next 6 months. Practices in the upper 50th percentile after the initial 6 months of intervention will continue to receive practice facilitation alone. The main outcome measures include the number (and %) of patients in the target population who are screened for unhealthy alcohol use, screen positive, and receive brief counseling. Additional measures include the number (and %) of patients who receive pharmacotherapy for AUD or are referred for AUD services. Sample size calculations determined that 35 practices are needed to detect a 10% increase in the main outcome (percent screened for unhealthy alcohol use) over 6 months. DISCUSSION: A successful intervention would significantly reduce morbidity among adults from unhealthy alcohol use by increasing counseling and other treatment opportunities. The study will produce important evidence about the effect of practice facilitation on uptake of evidence-based screening, counseling, and pharmacotherapy for unhealthy alcohol use when delivered on a large scale to small and medium-sized practices. It will also generate scientific knowledge about whether embedded telehealth services can improve the use of evidence-based screening and interventions for practices with slower uptake. The results of this rigorously conducted evaluation are expected to have a positive impact by accelerating the dissemination and implementation of evidence related to unhealthy alcohol use into primary care practices. TRIAL REGISTRATION: ClinicalTrials.gov NCT04317989 . Registered on March 23, 2020.

Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E.

The World Health Organization has designated Zika virus (ZIKV) as a dangerous, mosquito-borne pathogen that can cause severe developmental defects. The primary goal of this work was identification of small molecules as potential ZIKV inhibitors that target the viral envelope glycoprotein (ZIKV E) involved in membrane fusion and viral entry. A homology model of ZIKV E containing the small molecule ß-octyl glucoside (BOG) was constructed, on the basis of an analogous X-ray structure from dengue virus, and >4 million commercially available compounds were computationally screened using the program DOCK6. A key feature of the screen involved the use of similarity-based scoring to identify inhibitor candidates that make similar interaction energy patterns (molecular footprints) as the BOG reference. Fifty-three prioritized compounds underwent experimental testing using cytotoxicity, cell viability, and tissue culture infectious dose 50% (TCID50) assays. Encouragingly, relative to a known control (NITD008), six compounds were active in both the cell viability assay and the TCID50 infectivity assay, and they showed activity in a third caspase activity assay. In particular, compounds 8 and 15 (tested at 25 µM) and compound 43 (tested at 10 µM) appeared to provide significant protection to infected cells, indicative of anti-ZIKV activity. Overall, the study highlights how similarity-based scoring can be leveraged to computationally identify potential ZIKV E inhibitors that mimic a known reference (in this case BOG), and the experimentally verified hits provide a strong starting point for further refinement and optimization efforts.

Macrophages Infected by a Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Reveal Differential Reprogramming Signatures Early in Infection.

Despite their high degree of genomic similarity, different spotted fever group (SFG) Rickettsia are often associated with very different clinical presentations. For example, Rickettsia conorii causes Mediterranean spotted fever, a life-threatening disease for humans, whereas Rickettsia montanensis is associated with limited or no pathogenicity to humans. However, the molecular basis responsible for the different pathogenicity attributes are still not understood. Although killing microbes is a critical function of macrophages, the ability to survive and/or proliferate within phagocytic cells seems to be a phenotypic feature of several intracellular pathogens. We have previously shown that R. conorii and R. montanensis exhibit different intracellular fates within macrophage-like cells. By evaluating early macrophage responses upon insult with each of these rickettsial species, herein we demonstrate that infection with R. conorii results in a profound reprogramming of host gene expression profiles. Transcriptional programs generated upon infection with this pathogenic bacteria point toward a sophisticated ability to evade innate immune signals, by modulating the expression of several anti-inflammatory molecules. Moreover, R. conorii induce the expression of several pro-survival genes, which may result in the ability to prolong host cell survival, thus protecting its replicative niche. Remarkably, R. conorii-infection promoted a robust modulation of different transcription factors, suggesting that an early manipulation of the host gene expression machinery may be key to R. conorii proliferation in THP-1 macrophages. This work provides new insights into the early molecular processes hijacked by a pathogenic SFG Rickettsia to establish a replicative niche in macrophages, opening several avenues of research in host-rickettsiae interactions.

The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes.

Sphingolipid derivatives play key roles in immune cell migration and function. Synthetic sphingolipid analogues are used as therapeutics to intervene various inflammatory and malignant conditions. We hypothesize that different analogs have different effects on immune cells and therefore can be used as treatment for specific diseases. This study examines the properties of the novel synthetic sphingolipid analog, AD2900, and its effects on immune cell activation and lymphocyte localization in homeostasis. AD2900 is an antagonist for all sphingosine-1-phosphate (S1P) receptors. It demonstrates a significant inhibitory effect on the proliferation of activated human peripheral blood mononuclear cells, which is dependent on cAMP reduction and calcium signal transduction but not on phospholipase C activation. AD2900 causes a significant but reversible downregulation of S1P1 expression on the cell surface. AD2900 administration to C57BL/6J mice leads to the accumulation of T cells in the blood and spleen and in turn reduces T-cell number in the lymph nodes. Moreover, AD2900 treatment shows significant effects on the localization of T-cell subpopulations. These results demonstrate the key roles of S1P in T-cell trafficking in a steady state and suggest a potential clinical application for AD2900. Notably, this sphingolipid analog does not cause a severe lymphopenia. The clinical effect of AD2900 in hemato-oncologic diseases and immune-related diseases needs further investigation.

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells.

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

Crystal structure of phenyl 2,4,5-tri-chloro-benzene-sulfonate.

The title compound, C12H7Cl3O3S, was synthesized via a nucleophilic substitution reaction between phenol and 2,4,5-tri-chloro-benzene-sulfonyl chloride. The two aryl rings are oriented gauche to one another around the sulfonate S-O bond, with a C-S-O-C torsion angle of -70.68 (16)°, and the two rings are inclined to one another by 72.40 (7)°. In the crystal, mol-ecules are linked via various C-Cl⋯π inter-actions, forming ribbons propagating along [100]. Neighboring ribbons are linked by a weak C-Cl⋯π inter-action, forming layers parallel to (010).

A novel selective androgen receptor modulator (SARM) MK-4541 exerts anti-androgenic activity in the prostate cancer xenograft R-3327G and anabolic activity on skeletal muscle mass & function in castrated mice.

The androgen receptor (AR) is a member of the nuclear hormone receptor super family of transcription factors. Androgens play an essential role in the development, growth, and maintenance of male sex organs, as well as the musculoskeletal and central nervous systems. Yet with advancing age, androgens can drive the onset of prostate cancer, the second leading cause of cancer death in males within the United States. Androgen deprivation therapy (ADT) by pharmacologic and/or surgical castration induces apoptosis of prostate cells and subsequent shrinkage of the prostate and prostate tumors. However, ADT is associated with significant musculoskeletal and behavioral adverse effects. The unique pharmacological activity of selective androgen receptor modulator (SARM) MK-4541 recently has been reported as an AR antagonist with 5α-reductase inhibitor function. The molecule inhibits proliferation and induces apoptosis in AR positive, androgen dependent prostate cancer cells. Importantly, MK-4541 inhibited androgen-dependent prostate growth in male rats yet maintained lean body mass and bone formation following ovariectomy in female rats. In the present study, we evaluated the effects of SARM MK-4541 in the androgen-dependent Dunning R3327-G prostate carcinoma xenograft mouse model as well as on skeletal muscle mass and function, and AR-regulated behavior in mice. MK-4541 significantly inhibited the growth of R3327-G prostate tumors, exhibited anti-androgen effects on the seminal vesicles, reduced plasma testosterone concentrations in intact males, and inhibited Ki67 expression. MK-4541 treated xenografts appeared similar to xenografts in castrated mice. Importantly, we demonstrate that MK-4541 exhibited anabolic activity in androgen deficient conditions, increasing lean body mass and muscle function in adult castrated mice. Moreover, MK-4541 treatment restored general activity levels in castrated mice. Thus, MK-4541 exhibits an optimum profile as an adjuvant therapy to ADT which may provide potent anti-androgenic activity at the prostate yet protective activity on skeletal muscle and behavior in patients.

Crystal structure of 3,5-di-methyl-phenyl 2-nitro-benzene-sulfonate.

The title compound, C14H13NO5S, was synthesized via a nucleophilic substitution reaction between 3,5-di-methyl-phenol and 2-nitro-benzene-sulfonyl chloride. The aromatic rings attached to the SO3 group are oriented in a gauche fashion around the ester S-O bond, with a C-S-O-C torsion angle of 84.68 (11)°. The mol-ecules form centrosymmetric dimers via π-π stacking inter-actions between 3,5-di-methyl-phenyl groups (centroid-centroid distance = 3.709 Å). An inter-molecular S=O⋯N inter-action between the sulfonyl and nitro groups, with an O⋯N distance of 2.9840 (18) Å, organizes the dimers into columns extending along [011]. These columns are further assembled into (111) layers through C-H⋯O inter-actions.

Crystal structure of 2,4-di-nitro-phenyl 4-methyl-benzene-sulfonate: a new polymorph.

The title compound, C13H10N2O7S, was synthesized via a nucleophilic substitution reaction between 2,4-di-nitro-phenol and p-toluene-sulfonyl chloride. This crystal structure is a polymorph of CSD entry WUVYUH [Vembu et al. (2003). Acta Cryst, E59, o378-380]. The aromatic substituents on the sulfonate group are oriented gauche to one another with a C-O-S-C torsion angle of -62.0 (3)°. The supra-molecular features that contribute to the crystal stability are offset π-π [centroid-centroid distance = 3.729 (2) Å] and multiple C-H⋯O inter-actions.

Novel endosomolytic poly(amido amine) polymer conjugates for systemic delivery of siRNA to hepatocytes in rodents and nonhuman primates.

The application of small interfering (si)RNAs as potential therapeutic agents requires safe and effective methods for their delivery to the cytoplasm of the target cells and tissues. Recent studies have shown significant progress in the development of targeting reagents that facilitate the recognition of, and siRNA delivery to, specific cell types. Among recently reported delivery approaches, polymers with amphipathic properties have been used to enable endosome escape and cytosolic delivery. Here, we describe a linear amphipathic poly(amido amine) polymer conjugate system for the efficient siRNA delivery in vitro and in vivo. This polymer contains a novel amine bearing bis-acrylamide monomer designed for increasing amine density, which resulted in substantial improvement in liver uptake and RNAi activity compared to our previously reported poly(amido amine disulfide) polymer.1 The activity for this liver targeted delivery system was demonstrated in rodents and nonhuman primates.

An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

The Rickettsia conorii autotransporter protein Sca1 promotes adherence to nonphagocytic mammalian cells.

The pathogenesis of spotted fever group (SFG) Rickettsia species, including R. conorii and R. rickettsii, is acutely dependent on adherence to and invasion of host cells, including cells of the mammalian endothelial system. Bioinformatic analyses of several rickettsia genomes revealed the presence of a cohort of genes designated sca genes that are predicted to encode proteins with homology to autotransporter proteins of Gram-negative bacteria. Previous work demonstrated that three members of this family, rOmpA (Sca0), Sca2, and rOmpB (Sca5) are involved in the interaction with mammalian cells; however, very little was known about the function of other conserved rickettsial Sca proteins. Here we demonstrate that sca1, a gene present in nearly all SFG rickettsia genomes, is actively transcribed and expressed in R. conorii cells. Alignment of Sca1 sequences from geographically diverse SFG Rickettsia species showed that there are high degrees of sequence identity and conservation of these sequences, suggesting that Sca1 may have a conserved function. Using a heterologous expression system, we demonstrated that production of R. conorii Sca1 in the Escherichia coli outer membrane is sufficient to mediate attachment to but not invasion of a panel of cultured mammalian epithelial and endothelial cells. Furthermore, preincubation of a recombinant Sca1 peptide with host cells blocked R. conorii cell association. Together, these results demonstrate that attachment to mammalian cells can be uncoupled from the entry process and that Sca1 is involved in the adherence of R. conorii to host cells.