Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system.

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver IFT140 , PCDH15 , CEP290 , and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, this approach significantly surpasses the trans-splicing and split-intein-based reconstitution methods in efficiency, requiring lower doses, minimizing or eliminating the production of truncated protein products, and offering flexibility in selecting splitting positions. The CRE-lox approach described here provides a simple and effective platform for producing AAV gene therapy vectors beyond AAV's packaging capacity.

The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-hRS1 in a mouse model of X-linked retinoschisis.

Purpose: X-linked retinoschisis (XLRS), due to loss-of-function mutations in the retinoschisin (RS1) gene, is characterized by a modest to severe decrease in visual acuity. Clinical trials for XLRS utilizing intravitreal (IVT) gene therapy showed ocular inflammation. We conducted a subretinal dose-response preclinical study using rAAV2tYF-CB-hRS1 utilizing the Rs1 knockout (Rs1-KO) mouse to investigate short- and long-term retinal rescue after subretinal gene delivery. Methods: Rs1-KO mice were subretinally injected with 2 µL of rAAV2tYF-CB-hRS1 vector with 8E9 viral genomes (vg)/eye, 8E8 vg/eye, 8E7 vg/eye, or sham injection, and compared to untreated eyes. Reconstitution of human RS1 protein was detected using western blotting. Analysis of retinal function by electroretinography (ERG) and structural analysis by optical coherence tomography (OCT) were performed at 1, 2, 3, 5, 7, and 12 months post injection (MPI). Immunohistochemistry (IHC) was performed to evaluate cone rescue on the cellular level. Functional vision was evaluated using a visually guided swim assay (VGSA). Results: Western blotting analysis showed human RS1 protein expression in a dose-dependent manner. Quantification of western blotting showed that the RS1 protein expression in mice treated with the 8E8 vg dose was near the wild-type (WT) expression levels. ERG demonstrated dose-dependent effects: At 1 MPI the 8E8 vg dose treated eyes had higher light-adapted (LA) ERG amplitudes in 3.0 flash and 5 Hz flicker compared to untreated (p < 0.0001) and sham-treated eyes (p < 0.0001) which persisted until the 12 MPI endpoint, consistent with improved cone function. ERG b-wave amplitudes were higher in response to dark-adapted (DA) 0.01 dim flash and 3.0 standard combined response (SCR) compared to sham-treated (p < 0.01) and untreated eyes (p < 0.001) which persisted until 3 MPI, suggesting short-term improvement of the rod photoreceptors. All injections, including sham-treated, resulted in a cyst severity score of 1 (no cavities), with significant reductions compared to untreated eyes up to 3 MPI (p < 0.05). The high and low dose groups showed inconsistent ERG improvements, despite reduced cyst severity, emphasizing the dose-dependent nature of gene augmentation's efficacy and the tenuous connection between cyst reduction and ERG improvement. IHC data showed a significant cone rescue in eyes treated with the 8E8 vg dose compared to sham-treated and untreated eyes. VGSA showed better functional vision in 8E8 vg dose treated mice. Eyes treated with the highest dose showed occasional localized degeneration in the outer nuclear layer. Conclusion: Our data suggest that a dose of 8E8 vg/eye subretinally improves retinal function and structure in the Rs1-KO mouse. It improves cone function, rod function, and reduces cyst severity. Sham treatment resolves schisis cysts, but 8E8 vg/eye is needed for optimal retinal electrical function rescue. These findings offer a promising path for clinical translation to human trials.

Pooled Analysis of Trauma Centers Better Predicts Risk Factors for Firearm Violence Reinjury.

INTRODUCTION: Many trauma centers use the first firearm injury admission as a reachable moment to mitigate reinjury. Understanding repeat firearm violence can be difficult in metropolitan areas with multiple trauma centers and laws that prohibit sharing private health information across health systems. We hypothesized that risk factors for repeat firearm violence could be better understood using pooled data from two major metropolitan trauma centers. METHODS: Two level I trauma center registries were queried (2007-2017) for firearm injury admissions using International Classification of Diseases, Ninth and Tenth Revision (ICD9/10) Ecodes. A pseudo encryption tool allowed sharing of deidentified firearm injury and repeat firearm injury data without disclosing private health information. Factors associated with firearm reinjury admissions including, age, sex, race, payor, injury severity, intent, and discharge, were assessed by multivariable logistic regression. RESULTS: We identified 2145 patients with firearm injury admissions, 89 of whom had a subsequent repeat firearm injury admission. Majority of repeat firearm admissions were assaulted (91%), male (97.8%), and non-Hispanic Black (86.5%). 31.5% of repeat firearm injury admissions were admitted to a different trauma center from their initial admission. Independent predictors of repeat firearm injuries were age (adjusted odds ratio [aOR] 0.94, P < 0.001), male sex (aOR 6.18, P = 0.013), non-Hispanic Black race (aOR 5.14, P = 0.007), or discharge against medical advice (aOR 6.64, P=<0.001). CONCLUSIONS: Nearly a third of repeat firearm injury admissions would have been missed in the current study without pooled metropolitan trauma center data. The incidence of repeat firearm violence is increasing and those at the highest risk for reinjury need to be targeted for mitigating interventions.

A visually guided swim assay for mouse models of human retinal disease recapitulates the multi-luminance mobility test in humans.

PURPOSE: The purpose of this study was to develop a visually guided swim assay (VGSA) for measuring vision in mouse retinal disease models comparable to the multi-luminance mobility test (MLMT) utilized in human clinical trials. METHODS: Three mouse retinal disease models were studied: Bardet-Biedl syndrome type 1 (Bbs1M390R/M390R), n = 5; Bardet-Biedl syndrome type 10 (Bbs10-/-), n = 11; and X linked retinoschisis (retinoschisin knockout; Rs1-KO), n = 5. Controls were normally-sighted mice, n = 10. Eyeless Pax6Sey-Dey mice, n = 4, were used to determine the performance of animals without vision in VGSA. RESULTS: Eyeless Pax6Sey-Dey mice had a VGSA time-to-platform (TTP) 7X longer than normally-sighted controls (P < 0.0001). Controls demonstrated no difference in their TTP in both lighting conditions; the same was true for Pax6Sey-Dey. At 4-6 M, Rs1-KO and Bbs10-/- had longer TTP in the dark than controls (P = 0.0156 and P = 1.23 × 10-8, respectively). At 9-11 M, both BBS models had longer TTP than controls in light and dark with times similar to Pax6Sey-Dey (P < 0.0001), demonstrating progressive vision loss in BBS models, but not in controls nor in Rs1-KO. At 1 M, Bbs10-/- ERG light-adapted (cone) amplitudes were nonrecordable, resulting in a floor effect. VGSA did not reach a floor until 9-11 M. ERG combined rod/cone b-wave amplitudes were nonrecordable in all three mutant groups at 9-11 M, but VGSA still showed differences in visual function. ERG values correlate non-linearly with VGSA, and VGSA measured the continual decline of vision. CONCLUSION: ERG is no longer a useful endpoint once the nonrecordable level is reached. VGSA differentiates between different levels of vision, different ages, and different disease models even after ERG is nonrecordable, similar to the MLMT in humans.

Solid-state mechanochemical synthesis of chitosan from mud crab (Scylla serrata) chitin.

This study presents a method for solvent-free mechanochemical synthesis of chitosan from chitin, sourced from the shells of mud crabs (Scylla serrata). The procedure involves a sequence of demineralization and deproteinization to extract chitin from the crab shells, followed by mechanochemical deacetylation. The chitin was deacetylated by grinding it as a solid blend with sodium hydroxide (NaOH) using a stainless steel mortar and pestle. After grinding, chitosan is isolated from the blend by repetitive washing and centrifugation. The chitosan product is then characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction analysis. These characterization techniques confirm the successful deacetylation of chitin to form chitosan. A high degree of deacetylation (DD) is achieved when the weight ratio of NaOH to chitin is 1:1 or higher, implying that the DD value can be enhanced by increasing this weight ratio. The mechanochemical reaction mechanism involves the hydroxyl groups on the NaOH particles reacting with the acetamide groups of the chitin strands, yielding solid chitosan and sodium acetate. This mechanochemical deacetylation approach is more practical than the conventional heterogeneous deacetylation in strong basic solutions, since it could suppress depolymerization of the resulting chitosan and requires significantly less base. This makes it a promising method for large-scale industrial applications.

Portulaca oleracea L. (purslane) improves the anti-inflammatory, antioxidant and autophagic actions of metformin in the hippocampus of diabetic demented rats.

Great body of evidence links cognitive decline to diabetes/insulin resistance. In this study the effect of Portulaca oleracea (PUR) (100 mg/kg), Metformin (MET) (200 mg/kg), a first line diabetes mellitus type 2 therapy, and their combination on cognitive function and hippocampal markers in diabetic rats were assessed. Male rats were injected with streptozotocin (30 mg/kg on two successive weeks) followed by 4 weeks of treatment. Possible antioxidant, anti-inflammatory, and autophagy enhancing mechanisms of these drugs were investigated in the hippocampal tissue using spectrophotometry, ELISA, and western blotting. Diabetic rats suffered significant cognitive impairment in Morris's water maze, hippocampal TBARS elevation, GSH depletion, and SOD upregulation. In addition, diabetes promoted the secretion of hippocampal inflammatory cytokines, TNF-α and IL-1ß, and depleted anti-inflammatory cytokines as IL-10. Such detrimental changes were reversed by MET and/or PUR. Notably, AMPK was upregulated by diabetes, then restored to normal by MET and/or PUR. The pattern of change in AMPK expression was concomitant with changes in oxidative and inflammatory burden. Hence, AMPK is believed to be a key mediator in most of the measured pre-AD markers in this study. However, from our results, PUR is believed to have non-AMPK dependent actions as well. In conclusion, antidiabetic agents as metformin and purslane extract proved to be invaluable in addressing the cognitive decline and hippocampal changes that arise as a complication of diabetes. They mainly acted through AMPK pathway; however, their usefulness was not limited to AMPK pathways since their combination was suggested to have a different mechanism.

Use of 4-D proteomics to differentiate between bovine and camel lactoferrin.

Lactoferrin is a multifunctional protein that has various biological activities and applications. However, different sources of lactoferrin may have different properties and characteristics. In this study, we hypothesized that ultra-performance liquid chromatography quadrupole time-of-flight mass spectroscopy (UPLC-QTOF-IMS) coupled with UNIFI software can differentiate bovine lactoferrin from camel lactoferrin based on the unique peptides produced by trypsin digestion. We enzymatically digested the proteins using trypsin and analyzed the resulting peptides using Uniport software and in silico digestion. We identified 14 marker peptides that were unique to bovine lactoferrin and could be used to distinguish it from camel lactoferrin. We also demonstrated the advantages of 4D proteomics over 3D proteomics in separating and identifying peptides based on their mass, retention time, intensity, and ion mobility. This method can be applied to other lactoferrin sources and improve the quality control and authentication of lactoferrin products.

Nanoparticles of ZnO/Berberine complex contract COVID-19 and respiratory co-bacterial infection in addition to elimination of hydroxychloroquine toxicity.

Purpose: A novel coronavirus (COVID-19) that has not been previously identified in humans and has no specific treatment has recently spread. Treatment trials using antiviral and immune-modulating drugs such as hydroxychloroquine (HCQ) were used to control this viral outbreak however several side effects have emerged. Berberine (BER) is an alkaloid that has been reported to reveal some pharmacological properties including antioxidant and antimicrobial activities. Additionally, Zinc oxide nanoparticles (ZnO-NPs) possess potent antioxidant and anti-inflammatory properties. Therefore, this study was undertaken to estimate the efficiency of both BER and synthetic ZnO/BER complex as an anti-COVID-19 therapy. Methods: First, the ZnO/BER complex was prepared by the facile mixing method. Then in vitro studies on the two compounds were conducted including VeroE6 toxicity, anti-COVID-19 activity, determination of inhibitory activity towards papain-like proteinase (PL pro) and spike protein- and receptor- binding domain (RBD) as well as assessment of drug toxicity on RBCs. Results: The results showed that ZnO/BER complex acts as an anti-COVID-19 by inhibiting spike protein binding with angiotensin-converting enzyme II (ACE II), PL pro activity, spike protein and E protein levels, and expression of both E-gene and RNA dependent RNA polymerase (RdRp) at a concentration lower than that of BER or ZnO-NPs alone. Furthermore, ZnO/BER complex had antioxidant and antimicrobial properties where it prevents the auto oxidation of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and the culture of lower respiratory system bacteria that affected Covid 19 patients. The ZnO/BER complex prevented as well the HCQ cytotoxic effect on both RBC and WBC (in vitro) and hepatotoxicity, nephrotoxicity and anemia that occurred after HCQ long administration in vivo. Conclusion: The ZnO/BER complex can be accounted as promising anti-COVID 19 candidate because it inhibited the virus entry, replication, and assembly. Furthermore, it could be used to treat a second bacterial infection that took place in hospitalized COVID 19 patients. Moreover, ZnO/BER complex was found to eliminate the toxicity of long-term administration of HCQ in vivo.

Potential therapeutic and pharmacological strategies for SARS-CoV2.

BACKGROUND: At the end of 2019, the new Coronavirus disease 2019 (COVID-19) strain causing severe acute respiratory syndrome swept the world. From November 2019 till February 2021, this virus infected nearly 104 million, with more than two million deaths and about 25 million active cases. This has prompted scientists to discover effective drugs to combat this pandemic. AREA COVERED: Drug repurposing is the magic bullet for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). Therefore, several drugs have been investigated in silico, in vitro, as well as through human trials such as anti-SARS-CoV2 agents, or to prevent the complications resulting from the virus. In this review, the mechanisms of action of different therapeutic strategies are summarized. According to the WHO, different classes of drugs can be used, including anti-malarial, antiviral, anti-inflammatory, and anti-coagulant drugs, as well as angiotensin-converting enzyme inhibitors, antibiotics, vitamins, zinc, neutralizing antibodies, and convalescent plasma therapy. Recently, there are some vaccines which are approved against SARS-CoV2. EXPERT OPINION: A complete understanding of the structure and function of all viral proteins that play a fundamental role in viral infection, which contribute to the therapeutic intervention and the development of vaccine in order to reduce the mortality rate. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40005-021-00520-4.

Knowledge and Attitude Regarding Toxoplasmosis among Jazan University Female Students.

BACKGROUND: In Saudi Arabia, the prevalence of toxoplasmosis is high. However, to date, few studies have evaluated the degree of knowledge on toxoplasmosis among females in Saudi Arabia. OBJECTIVES: The objective of this study was to assess the knowledge, attitude and preventive behavior regarding toxoplasmosis among female students at Jazan University, Jazan, southwest Saudi Arabia. MATERIALS AND METHODS: This cross-sectional study was conducted on a random sample of 440 female students at Jazan University using a semi-structured, self-administered questionnaire. Data with numerical/qualitative variables were expressed as frequency and percentage. Chi-square test was used to analyze categorical variables. P < 0.05 was used to indicate statistical significance. RESULTS: This study found that more than three-quarters (79.1%) of the students had insufficient knowledge about toxoplasmosis. Students from healthcare faculties had higher knowledge scores (28.5%) than students from arts and humanities (20.6%) and science (18.9%) faculties; however, the differences were not statistically significant (P = 0.77). The majority of the studied sample (92.3%) was found to eat fast food on a regular basis. About 42%, 54% and 4% of the respondents reported that they never, occasionally and always ate improperly washed vegetables, respectively. CONCLUSIONS: This study found that a substantial proportion of Jazan University's female students have insufficient knowledge on toxoplasmosis. Health educational programs are necessary to increase the awareness and knowledge about toxoplasmosis and its clinical manifestations.

Comorbidities associated with obstructive sleep apnea: a retrospective Egyptian study on 244 patients.

PURPOSE: The aim of the present study was to assess prevalence of associated comorbidities in a group of patients diagnosed with obstructive sleep apnea syndrome (OSAS). METHODS: This retrospective study enrolled 244 consecutive patients diagnosed by polysomnogram with OSAS between October 2010 and January 2015 after being referred to our Sleep-Related Breathing Disorders Unit, Chest Diseases Department, in the Alexandria Main University Hospital. RESULTS: Of 244 patients, 47% were men, mean age was 56.9 years, and mean apnea-hypopnea index was 43.6 events per hour. Patients were categorized into two groups: group 1 (38%), mild and moderate OSAS, and group 2 (62%), severe, very severe, and extreme OSAS. Comorbidities were present in 91% of patients. The most common comorbidities were obesity, hypertension (HTN), and diabetes mellitus (DM). Prevalence of obesity, HTN, DM, congestive heart failure, deep vein thrombosis, pulmonary embolism (PE), and hypothyroidism was significantly higher in severity group 2. PE, bronchial asthma, and chronic obstructive pulmonary disease were significantly higher among men, whereas hypothyroidism was significantly higher among women. During this period of over 4 years, mortality rate was 8%. The majority of deaths occurred at night. Most of the studied patients (60%) either received no treatment or were not adherent to positive airway pressure (PAP) therapy. None of the patients received surgicaltreatment. The majority (50%) gained access to PAP therapy through donations. Associated hypoventilation was the only significant predictor of PAP adherence. Quality of life was significantly better among PAP adherent patients. CONCLUSIONS: Patients suffering from OSAS have very high prevalence of comorbidities indicating a great burden on the healthcare system. Despite this fact, over 50% of the patients studied did not receive any treatment. Charities were the main portal fortreatment.

Trigger Point Manual Therapy for the Treatment of Chronic Noncancer Pain in Adults: A Systematic Review and Meta-analysis.

OBJECTIVE: To determine the effectiveness of trigger point manual therapy (TPMT) for reducing chronic noncancer pain and associated problems in adults, by analyzing all relevant randomized controlled trials (RCTs). DATA SOURCES: We searched databases and clinical trials registers from their inception to May 2017. STUDY SELECTION: We included RCTs in any language that recruited patients older than 18, with pain of 3 months' duration or more. We assessed pain, function, and patient-reported improvement as outcomes. DATA EXTRACTION: Two authors independently extracted and verified data. Meta-analysis was completed where possible, otherwise data were synthesized narratively. DATA SYNTHESIS: We combined all data using a random-effects model and assessed the quality of evidence using GRADE. A total of 19 trials (involving 1047 participants) met inclusion criteria, representing TPMT treatment of musculoskeletal, pelvic, and facial pain. No effect was found for short-term pain relief (mean standardized difference -0.53; 95% confidence interval [CI], -1.08 to 0.02). One small study showed a longer-term benefit for pain (mean standardized difference -2.00; 95% CI, -3.40 to -0.60) but with low confidence in the effect. Significant gains emerged for function (mean standardized difference -0.77; 95% CI, -1.27 to -0.26) and in patient global response (odds ratio 3.79; 95% CI, 1.86-7.71) from 4 studies, but not for health-related quality of life. CONCLUSIONS: Evidence for TPMT for chronic noncancer pain is weak and it cannot currently be recommended.

Knowledge, attitude and practices of women in maldives related to the risk factors, prevention and early detection of cervical cancer.

BACKGROUND: A population-based cervical cancer screening program using visual inspection with acetic acid was launched in Maldives in 2014. Our study aimed to assess the knowledge, attitude and practices of women in relation to risk factors of cervical cancer, early detection of the disease and its prevention. MATERIALS AND METHODS: The questionnaire based survey was conducted among 20 to 50 year old women, systematically sampled to represent three regions of Maldives. Trained investigators interviewed a total of 2,845 women at home. RESULTS: The prevalence of the risk factors of cervical cancer like early age at marriage and childbirth, multiple marriages, multiple marriages of the husbands, and multiple pregnancies was high. More women knew about breast cancer than cervical cancer. Even among the small number of women who knew of cervical cancer, only 34.6% had the knowledge of at least one early symptom. Very few women knew that the cancer could be prevented by any test. Only 6.2% of the women reported having ever undergone a Pap smear. Many women had the misconception that cervical cancer was infectious. In Maldives the younger women have high literacy rate due to the policy of universal free education and those with higher levels of education had improved knowledge of cervical cancer and its risk factors. The prevalence of risk factors also reduced with improved literacy. CONCLUSIONS: Awareness about risk factors and prevention of cervical cancer is limited among Maldivian women in spite of having high exposure to some of the risk factors. A universal literacy program in the country has helped to improve the knowledge of cervical cancer prevention and to reduce the exposure to various risk factors in the younger population.