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It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

The increase in cardiovascular risk in patients with rheumatoid arthritis (RA) compared with the general population is due to the combined effect of traditional risk factors for cardiovascular diseases, metabolic disorders, systemic inflammation, and side effects of antirheumatic drugs. Tofacitinib (TOFA) is an oral reversible inhibitor of janus kinases for the treatment of RA with proven efficacy and good tolerability, but its effects on body weight and metabolic profile need to be clarified. We investigated the effects of TOFA on body mass index (BMI) and visceral adiposity index (VAI) in RA patients. Thirty-one consecutive patients with active RA and starting new treatment with TOFA were included in a prospective 1 year follow-up observational study of cardiovascular effects of TOFA treatment. Weight, height, waist circumference, BMI, blood pressure, lipid profile, fasting glucose and VAI were measured at baseline and 12 months of treatment. Median weight gain was 3 kg (4.2%) after 1 year of TOFA. 23 (74%) patients suffered from a weight gain, and 6 (26%) out of them from a weight increment of 10% or more. Patients with lower BMI (p = 0.024) and higher baseline DAS28 [ESR] (p = 0.017) have the risk of an increase in BMI > 5% during TOFA treatment in a multivariate analysis. A decrease in VAI after 12 months was recorded. Weight increment and improvement of VAI are frequent on TOFA treatment. BMI dynamics associated with higher disease activity at baseline and lower baseline BMI.

We evaluated changes in gene expression of mTOR, p21, caspase-3, ULK1, TNFα, matrix metalloproteinase (MMP)-9, and cathepsin K in the whole blood of rheumatoid arthritic (RA) patients treated with methotrexate (MTX) in relation to their rheumatoid factor status, clinical, immunological, and radiological parameters, and therapeutic response after a 24-month follow-up. The study group consisted of 35 control subjects and 33 RA patients without previous history of MTX treatment. Gene expression was measured using real-time RT-PCR. Decreased disease activity in patients at the end of the study was associated with significant downregulation of TNFα expression. Downregulation of mTOR was observed in seronegative patients, while no significant changes in the expression of p21, ULK1, or caspase-3 were noted in any RA patients at the end of the study. The increase in erosion numbers observed in the seropositive patients at the end of the follow-up was accompanied by upregulation of MMP-9 and cathepsin K, while seronegative patients demonstrated an absence of significant changes in MMP-9 and cathepsin K expression and no increase in the erosion score. Our results suggest that increased expression of MMP-9 and cathepsin K genes in the peripheral blood might indicate higher bone tissue destruction activity in RA patients treated with methotrexate. The clinical study registration number is 0120.0810610.

In orthopedic trauma surgery, spatially structured biofilm ecosystems of bacteria that colonize orthopedic devices account for up to 65% of all healthcare infections, including tens of millions of people affected in the United States. These biofilm infections typically show increased resistance to antibiotics due to their structure and composition, which contributes significantly to treatment failure. Anti-biofilm approaches are needed together with clinically usable microscopic-resolution imaging techniques for treatment efficacy assessment. Antimicrobial photodynamic therapy (aPDT) has been recently proposed to combat clinically relevant biofilms (chronic wound infections, dental biofilms, etc.) using photosensitizers excited with visible light to generate reactive oxygen species that can kill bacteria residing within pathogenic biofilms. We aim to assess the efficacy of this treatment for eradication of biofilms typically present on surfaces of orthopedic devices (e.g., intramedullary nails and osseointegrated prosthetic implants). In the first phase reported here, we test aPDT by growing biofilms of and bacteria (two of the seven most common pathogens found in orthopedic trauma patients) inside soft lithography-fabricated microfluidic devices. We treat these biofilms with 5-aminolevulinic acid (5-ALA)-based aPDT, evaluate treatment efficacy with optical coherence tomography, and compare with regular clinical antibiotic treatment outcomes. The antibacterial efficiency of 5-ALA-based aPDT showed nonlinear dependence on the photosensitizer concentration and the light power density, with low parameters ( light dose, 5-ALA concentration) being significantly more effective than antibiotic-treated groups ( ), reaching 99.98% of bacteria killed at light dose and 5-ALA concentration setting. Performed experiments enable the translation of this portable treatment/imaging platform to the second phase of the study: aPDT treatment response assessment of biofilms grown on orthopedic hardware.

Orthopedic implant-associated infections cause serious complications primarily attributed to bacterial biofilm formation and are often characterized by increased antibiotic resistance and diminished treatment response. Yet, no methods currently exist to identify biofilms intraoperatively-surgeons rely solely on their eyes and hands and cannot detect or differentiate infected tissue to determine the location and extent of contamination. As the first step in addressing this unmet clinical need, here, we develop an optical coherence tomography (OCT)-based imaging method capable of detection and quantification of one of the most dangerous orthopedic biofilms formed by methicillin-resistant (MRSA). Growing biofilms on orthopedic hardware, we identify MRSA distinct optical signature through histogram-based multi-parametric texture analysis of OCT images and support the findings with bioluminescence imaging and scanning electron microscopy. Under identical experimental conditions, we identify an optical signature of ( ) biofilms and use it to distinguish and quantify both species within MRSA- biofilms. The developed OCT-based methodology was successfully tested for (1) MRSA colonies delineation, (2) detection of metal hardware (an important feature for clinical translation where the metal surface of most orthopedic hardware is not flat), (3) automated quantification of biofilm thickness and roughness, and (4) identification of pores and, therefore, ability to evaluate the role of porosity-one of the critical biological metrics in relation to biofilm maturity and response to treatment. For the first time, we demonstrated complex pore structures of thick ( ) MRSA biofilms with an unprecedented level of detail. The proposed rapid noninvasive detection/quantification of MRSA biofilms on metal surfaces and delineation of their complex network of pores opens new venues for label-free MRSA detection in preclinical models of trauma surgery, expansion to other bacterial strains, and further clinical translation.

Methicillin-resistant (MRSA) biofilm infections present a critical challenge in orthopedic trauma surgery and are notoriously resistant to systemic antibiotic therapy. Noninvasive, quantitative imaging methods are urgently needed to assess biofilm burden and therapeutic efficacy, especially for emerging photodynamic therapy (PDT) strategies. We aim to establish a quantitative framework using a combined bioluminescence and optical coherence tomography (OCT) imaging approach to correlate bioluminescent signal with viable MRSA burden in both planktonic and biofilm states and to determine how biofilm density and structure influence this relationship. Bioluminescent MRSA (SAP231-luxCDABE) was cultured in planktonic and biofilm forms using growth models in 24-well plates and custom macrofluidic devices, respectively. Bacteria bioluminescence intensity (BLI), counted colony-forming units (CFU), and OCT-based biofilm thickness measurements were collected to construct linear regression models to evaluate how well BLI alone, or combined with biofilm density (CFU/volume), predicts bacterial counts across culture conditions. Bioluminescence strongly correlated with CFU in planktonic cultures ( ). In biofilms, BLI per CFU decreased with density, indicating metabolic downregulation, and BLI alone was less reliable ( ). Incorporating biofilm density (CFU/volume) improved prediction ( ). A joint model for both states showed excellent fit ( ), but the biofilm versus planktonic group remained a significant factor ( ), revealing systematic differences. This highlights the need for a mixed-model approach that segments subvolumes by morphological features to improve accurate, generalizable CFU estimation across both growth states. Bioluminescence alone underestimates bacterial burden in dense, metabolically suppressed MRSA biofilms. The combination of BLI with OCT-derived structural metrics enables accurate, nondestructive quantification of viable bacterial load. This approach provides a robust toolset for preclinical evaluation of antimicrobial therapies, particularly for optimizing PDT dosimetry and assessing biofilm response in translational infection models.

El propósito del artículo es desarrollar el paradigma educativo de la cultura de la gestión ambiental para una educación avanzada orientada al desarrollo sostenible. Se establecen los fundamentos metodológicos de la investigación, presentados por enfoques metodológicos culturales-ecológicos, axiológicos, de actividad personal, integrales-situacionales. Se definen los principales métodos de investigación: análisis sistémico-estructural, generalización, sistematización, diseño y experimentación pedagógica. Se revelan los criterios, indicadores y métodos para diagnosticar la formación de los componentes de la cultura de gestión de la naturaleza. A partir de la investigación se desarrolló una metodología para la formación de una cultura de gestión ambiental en el contexto de la educación no formal, que incluye el objetivo, el contenido, las etapas y la tecnología de las situaciones integrales de orientación cultural. Para implementar esta metodología en la práctica educativa, se desarrolló un programa de actividades para “equipos verdes” escolares: “Estamos en el paisaje cultural de las fincas de nuestra tierra natal”, se revela su objetivo, contenido, se muestra la interconexión de las secciones del programa con la tecnología de situaciones integrales culturalmente orientadas. Se presentan los resultados de un experimento pedagógico organizado en los sitios experimentales de la Universidad de Minin. Los resultados del experimento, en el que participaron 360 estudiantes de secundaria, permitieron sacar una conclusión sobre la eficacia de la formación de una cultura de gestión ambiental entre los estudiantes en el contexto de la educación no formal de orientación cultural y ecológica.