Liquid Biopsy Response Evaluation Criteria in Solid Tumors (LB-RECIST).

Current evaluation of treatment response in solid tumors depends on dynamic changes in tumor diameters as measured by imaging. However, these changes can only be detected when there are enough macroscopic changes in tumor volume, which limits the usability of radiological response criteria in evaluating earlier stages of disease response and necessitates much time to lapse for gross changes to be notable. One promising approach is to incorporate dynamic changes in circulating tumor DNA (ctDNA), which occur early in the course of therapy and can predict tumor responses weeks before gross size changes manifest. However, several issues need to be addressed before recommending the implementation of ctDNA response criteria in daily clinical practice such as clinical, biological, and regulatory challenges and, most importantly, the need to standardize/harmonize detection methods and ways to define ctDNA response and/or progression for precision oncology. Herein, we review the use of liquid biopsy (LB) to evaluate response in solid tumors and propose a plan toward standardization of LB-RECIST.

Association between anaesthesia-surgery team sex diversity and major morbidity.

BACKGROUND: Team diversity is recognized not only as an equity issue but also a catalyst for improved performance through diversity in knowledge and practices. However, team diversity data in healthcare are limited and it is not known whether it may affect outcomes in surgery. This study examined the association between anaesthesia-surgery team sex diversity and postoperative outcomes. METHODS: This was a population-based retrospective cohort study of adults undergoing major inpatient procedures between 2009 and 2019. The exposure was the hospital percentage of female anaesthetists and surgeons in the year of surgery. The outcome was 90-day major morbidity. Restricted cubic splines were used to identify a clinically meaningful dichotomization of team sex diversity, with over 35% female anaesthetists and surgeons representing higher diversity. The association with outcomes was examined using multivariable logistic regression. RESULTS: Of 709 899 index operations performed at 88 hospitals, 90-day major morbidity occurred in 14.4%. The median proportion of female anaesthetists and surgeons was 28 (interquartile range 25-31)% per hospital per year. Care in hospitals with higher sex diversity (over 35% female) was associated with reduced odds of 90-day major morbidity (OR 0.97, 95% c.i. 0.95 to 0.99; P = 0.02) after adjustment. The magnitude of this association was greater for patients treated by female anaesthetists (OR 0.92, 0.88 to 0.97; P = 0.002) and female surgeons (OR 0.83, 0.76 to 0.90; P < 0.001). CONCLUSION: Care in hospitals with greater anaesthesia-surgery team sex diversity was associated with better postoperative outcomes. Care in a hospital reaching a critical mass with over 35% female anaesthetists and surgeons, representing higher team sex-diversity, was associated with a 3% lower odds of 90-day major morbidity.

Analysis of LRBA pathogenic variants and the association with functional protein domains and clinical presentation.

LRBA is a cytoplasmic protein that is ubiquitously distributed. Almost all LRBA domains have a scaffolding function. In 2012, it was reported that homozygous variants in LRBA are associated with early-onset hypogammaglobulinemia. Since its discovery, more than 100 pathogenic variants have been reported. This review focuses on the variants reported in LRBA and their possible associations with clinical phenotypes. In this work LRBA deficiency cases reported more than 11 years ago have been revised. A database was constructed to analyze the type of variants, age at onset, clinical diagnosis, infections, autoimmune diseases, and cellular and immunoglobulin levels. The review of cases from 2012 to 2023 showed that LRBA deficiency was commonly diagnosed in patients with a clinical diagnosis of Common Variable Immunodeficiency, followed by enteropathy, neonatal diabetes mellitus, ALPS, and X-linked-like syndrome. Most cases show early onset of presentation at <6 years of age. Most cases lack protein expression, whereas hypogammaglobulinemia is observed in half of the cases, and IgG and IgA levels are isotypes reported at low levels. Patients with elevated IgG levels exhibited more than one autoimmune manifestation. Patients carrying pathogenic variants leading to a premature stop codon show a severe phenotype as they have an earlier onset of disease presentation, severe autoimmune manifestations, premature death, and low B cells and regulatory T cell levels. Missense variants were more common in patients with low IgG levels and cytopenia. This work lead to the conclusion that the type of variant in LRBA has association with disease severity, which leads to a premature stop codon being the ones that correlates with severe disease.

Laser beam shaping using a photoinduced azopolymer droplet-based mask.

The dewetting of an azopolymer droplet, followed by the photostructuration of the evaporated droplet, is employed to create an amplitude mask. This straightforward process yields a large area featuring periodic micro- and nanostructures. The resulting pattern is utilized to generate a nondiffracting beam. Starting with a Gaussian beam illuminating the amplitude mask, the critical aspect is the production of a bright, ring-shaped beam with a high radius-to-width ratio and symmetric central laser spots, each with the same intensity. This alternative approach to shaping a laser beam is demonstrated as a rapid and cost-effective fabrication technique.

The Association Between Hospital High-volume Anesthesiology Care and Patient Outcomes for Complex Gastrointestinal Cancer Surgery: A Population-based Study.

OBJECTIVE: To examine the association of between hospital rates of high-volume anesthesiology care and of postoperative major morbidity. BACKGROUND: Individual anesthesiology volume has been associated with individual patient outcomes for complex gastrointestinal cancer surgery. However, whether hospital-level anesthesiology care, where changes can be made, influences the outcomes of patients cared at this hospital is unknown. METHODS: We conducted a population-based retrospective cohort study of adults undergoing esophagectomy, pancreatectomy, or hepatectomy for cancer from 2007 to 2018. The exposure was hospital-level adjusted rate of high-volume anesthesiology care. The outcome was hospital-level adjusted rate of 90-day major morbidity (Clavien-Dindo grade 3-5). Scatterplots visualized the relationship between each hospital's adjusted rates of high-volume anesthesiology and major morbidity. Analyses at the hospital-year level examined the association with multivariable Poisson regression. RESULTS: For 7893 patients at 17 hospitals, the rates of high-volume anesthesiology varied from 0% to 87.6%, and of major morbidity from 38.2% to 45.4%. The scatter plot revealed a weak inverse relationship between hospital rates of high-volume anesthesiology and of major morbidity (Pearson: -0.23). The adjusted hospital rate of high-volume anesthesiology was independently associated with the adjusted hospital rate of major morbidity (rate ratio: 0.96; 95% CI, 0.95-0.98; P <0.001 for each 10% increase in the high-volume rate). CONCLUSIONS: Hospitals that provided high-volume anesthesiology care to a higher proportion of patients were associated with lower rates of 90-day major morbidity. For each additional 10% patients receiving care by a high-volume anesthesiologist at a given hospital, there was an associated reduction of 4% in that hospital's rate of major morbidity.

Enhanced passive surveillance for early detection of African and classical swine fevers.

African swine fever (ASF) and classical swine fever (CSF) are transboundary animal diseases (TADs) of pigs. Much effort and resources are regularly put into preventing these diseases' introduction in free areas. Passive surveillance activities bring the highest chances for the early detection of TAD incursions because they are routinely and widely conducted at farms, and because these activities focus on the time between introduction and when the first sample is sent for diagnostic testing. The authors proposed the implementation of an enhanced passive surveillance (EPS) protocol based on collecting data through participatory surveillance actions using an objective and adaptable scoring system to aid the early detection of ASF or CSF at the farm level. The protocol was applied in two commercial pig farms for ten weeks in the Dominican Republic, which is a CSF- and ASF-infected country. This study was a proof of concept, based on the EPS protocol to aid detection of substantial variations in the risk score triggering testing. One of the followed farms had score variation, which triggered testing of the animals, although the test results were negative. The study enables assessment of some of the weaknesses associated with passive surveillance and provides lessons applicable to the problem. Results demonstrate the potential for overcoming some issues preventing the broad application of EPS protocols and suggest that standardised approaches may contribute to the early detection of CSF and ASF introductions.

La peste porcine africaine (PPA) et la peste porcine classique (PPC) sont des maladies animales transfrontalières touchant les porcs. De nombreux efforts et ressources sont régulièrement alloués pour prévenir l'introduction de ces maladies dans des zones indemnes. Les activités de surveillance passive offrent les meilleures perspectives de détection précoce des incursions de maladies animales transfrontalières parce qu'elles sont menées de manière systématique et exhaustive dans les élevages, et parce qu'elles se concentrent sur la période entre l'introduction de la maladie et le moment où le premier échantillon est envoyé au laboratoire pour analyse. Les auteurs proposent la mise en oeuvre d'un protocole de surveillance passive renforcée fondé sur la collecte de données via des actions de surveillance participative utilisant un système de notation objectif et adaptable, en vue d'une détection précoce de la PPA et de la PPC dans les élevages. Ce protocole a été appliqué en République dominicaine, pays infecté par la PPA et la PPC, dans deux élevages porcins commerciaux pendant dix semaines. Cette étude était destinée à valider le principe de la méthode et se fondait sur le protocole de surveillance passive renforcée pour mieux détecter les variations substantielles de la note de risque qui conduisent à tester les animaux. L'un des élevages suivis a présenté une variation de cette note, ce qui a conduit à tester les animaux mais les tests se sont révélés négatifs. L'étude permet d'évaluer certaines des faiblesses associées à la surveillance passive et apporte des enseignements applicables à ce problème. Les résultats illustrent le potentiel de l'approche à surmonter certaines des problématiques empêchant l'application extensive des protocoles de surveillance passive renforcée. Ils suggèrent également que des approches normalisées pourraient contribuer à la détection précoce des cas d'introduction de la PPC et de la PPA.

La peste porcina africana (PPA) y la peste porcina clásica (PPC) son enfermedades animales transfronterizas que afectan al cerdo. Periódicamente se dedican grandes esfuerzos y cuantiosos recursos a evitar que estas enfermedades penetren en zonas que están exentas de ellas. Las actividades de vigilancia pasiva son las más eficaces para detectar con prontitud toda incursión de enfermedades animales transfronterizas, no solo por la regularidad y amplitud con que se llevan a cabo en las explotaciones, sino también porque inciden específicamente en el intervalo entre la penetración de una enfermedad y el momento en que se envía la primera muestra para que sea sometida a pruebas de diagnóstico. Los autores propusieron que se aplicara un protocolo de vigilancia pasiva reforzada que reposaba en la obtención de datos mediante actividades de vigilancia participativa, empleando para ello un sistema objetivo y adaptable de puntuación que ayudaba a detectar con prontitud la presencia en las explotaciones de PPA o PPC. Dicho protocolo fue aplicado a lo largo de diez semanas en dos explotaciones porcinas industriales de la República Dominicana, país en el que ambas infecciones están presentes. El estudio, que sirvió para poner a prueba la idea, pasaba por la aplicación del protocolo de vigilancia pasiva reforzada para ayudar a detectar variaciones sustanciales de la puntuación del nivel de riesgo que activa la realización de pruebas. En una de las explotaciones estudiadas se produjo una variación de la puntuación, cosa que activó la realización de pruebas en los animales, aunque estas arrojaron resultado negativo. El estudio aquí descrito permite evaluar algunos de los puntos débiles de la vigilancia pasiva y extraer enseñanzas aplicables al problema. Los resultados demuestran que es posible salvar algunas de las dificultades que impiden la aplicación generalizada de protocolos de vigilancia pasiva reforzada y dejan pensar que quizá el uso de planteamientos normalizados pueda ayudar a detectar con prontitud los casos de penetración de PPC o PPA.

Gingival neoplasms: a multicenter collaborative study of 888 patients in Brazil.

BACKGROUND: To evaluate the prevalence and clinicopathological features of a large series of gingival neoplasms in Brazil. MATERIAL AND METHODS:  All gingival benign and malignant neoplasms were retrieved from the records of six Oral Pathology Services in Brazil, during a 41-year period. Clinical and demographic data, clinical diagnosis, and histopathological data were collected from the patients' clinical charts. For statistical analysis, the chi-square, median test of independent samples and the U Mann-Whitney tests were used, considering a significance of 5%. RESULTS:  From 100,026 oral lesions, 888 (0.9%) were gingival neoplasms. There were 496 (55.9%) males, with a mean age of 54.2 years. Most cases (70.3%) were malignant neoplasms. Nodules (46.2%) and ulcers (38.9%) were the most common clinical appearance for benign and malignant neoplasms, respectively. Squamous cell carcinoma (55.6%) was the most common gingival neoplasm, followed by squamous cell papilloma (19.6%). In 69 (11.1%) malignant neoplasms, the lesions were clinically considered to be inflammatory or of infectious origin. Malignant neoplasms were more common in older men, appeared with larger size, and with a time of complaint shorter than benign neoplasms (p<0.001). CONCLUSIONS:  Benign and malignant tumors may appear as nodules in gingival tissue. In addition, malignant neoplasms, especially squamous cell carcinoma, should be considered in the differential diagnosis of persistent single gingival ulcers.

Possible zoonotic implications of the discovery of the advanced third stage larva of Gnathostoma turgidum (Spirurida: Gnathostomatidae) in a Mexican fish species.

Gnathostomiasis in humans is acquired by consumption of any infected second intermediate host or paratenic host. This includes amphibians, snakes and poultry as well as fish. In this work we report for the first time in Mexico the presence of an AdvL3 of Gnathostoma turgidum in the musculature of a wild fish (Gobiomorus dormitor, which also acts as intermediate host for the larvae of G. binucleatum and G. lamothei), from the Papaloapan River, Veracruz; previously, larvae of G. turgidum had only been recorded in amphibians in Mexico and in wild swamp eels from Tampa, Florida, USA. The larva found is extremely small (approximately 1,500 by 140 microns in length and width, respectively), and was obtained by artificial digestion with pepsin after examining the musculature against the light between two glass plates, a method by which it went unnoticed. Our finding of an AdvL3 in this fish, together with a previous molecular phylogenetic analysis revealing that the five species involved in human infections do not nest in the same clade, suggest that all species in the genus are potentially zoonotic. In this context, we strongly recommend the identification of larvae extracted from human patients at specific level, in order to know the role played by the 3 species distributed in Mexico in human cases of gnathostomiasis.

Prevalence of flukes (Fasciola hepatica and paramphistomids) in cattle in south-eastern Mexico.

The objective of this study was to determine the risk factors and prevalence of trematodes in south-eastern Mexico. The prevalence of trematodes was determined in 1010 bovines. The study was carried out from October 2018 (n=291) to December 2019 (n=719). Only in 2019 rumen and liver fluke eggs were differentiated. Faecal samples (n=311) were obtained from farms in southeast Mexico located in Tabasco, Chiapas and Campeche. In addition, the presence of flukes in liver and rumen from slaughtered cattle in abattoirs was recorded with a total of 408 samples. A logistic procedure was used to obtain the prevalence and the effect of main risk factors such as land physiography (flooded areas and hills), year, sex, animals' age and type of sample obtained (eggs in faeces and flukes). The general prevalence of flukes in cattle was 32.3 % in 2018 and 41.7 % in 2019. Prevalence of F. hepatica (liver fluke) was 18.6 % (134/719) and that of paramphistomids (rumen fluke) was 33.4 % (240/719). The infected cattle from the slaughterhouse indicated a lower prevalence of F. hepatica (1 %) and rumen fluke (26.7 %) than in farms detected by egg in faeces (41.8 % and 42.1 %, respectively). The physiographic zone was decisive in the presence of F. hepatica and rumen fluke, while sex did not represent a risk factor (P > 0.05). The environmental conditions of the Mexican southeast favour the presence of both liver and rumen fluke.

Uricaemia and associated health determinants in a paediatric population in Mexico.

BACKGROUND AND AIM: Uric acid (UA) is a product of the catabolism of purines, and its increase in blood may be related to the development of cardiometabolic diseases. Whether UA is the result or causal determinant of the appearance of risk factors for cardiometabolic disease is not yet known. UA levels among the young student population in San Luis Potosi have increased in recent years, which may be indicative of a serious future public health concern. Therefore, the objective of this study was to evaluate the association of sociodemographic, lifestyle and cardiometabolic determinants with UA levels in children and adolescents in San Luis Potosí. METHODS AND RESULTS: A total of 730 students (54.1% female and 45.9% male, 6-19 years old) participated in the study. The subjects attended one of five public schools located in San Luis Potosí. Venous blood samples were collected, blood serum was separated by centrifugation, and UA concentrations were measured with an automated analytical platform. UA was associated with most of the independent variables studied. It presented a positive correlation with body mass index (r = 0.363, p < 0.01). Male sex, socioeconomic status, total screen time, exercise, adequate sleep, systolic blood pressure, total cholesterol, and high-density lipoprotein cholesterol explained 23%-39% (p < 0.001) of the variability of plasma concentrations of UA in children and adolescents. CONCLUSION: Early detection of these determinants will prevent future diseases. Moreover, it will help with the implementation of preventive strategies that could improve the health of this population.

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins.

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of ß-lactoglobulin (ß-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for ß-LG (14% wt/wt), ß-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).

Altered phagocytic capacity due to acute exposure and long-term post-exposure to pesticides used for vector-borne disease as dengue.

Spinosad and temefos are widely used pesticides for chemical control of dengue vector-borne disease (Aedes aegypti). The aim of this study was to compare the effect of acute exposure (7 days) to spinosad (0.5 mg A.I. L-1) and temefos (10 mg A.I. L-1), concentrations used by the Mexican Ministry of Health, on phagocytic capacity (PC) of mononuclear cells of guppies fish (Poecilia reticulata), as well as to assess PC in fish, at 96 days after exposure to those pesticides. Obtained results indicated that spinosad did not alter PC, while an acute exposure to temefos significantly affected phagocytosis and this parameter was maintained downed even 96 days after the acute exposure, suggesting that the immunotoxic effects of temefos may be chronic.

Death of guppy fish (Poecilia reticulata) leukocytes induced by in vivo exposure to temephos and spinosad.

Temephos and spinosad are pesticides used for control of vector-borne diseases such as dengue, chikungunya and zika. However, the inadequate use of these substances has affected the health of non-target organisms. The aim of this study was to evaluate and compare, the effects of temephos and spinosad on leukocyte viability and death, using guppy fish (Poecilia reticulate) as a model organism. Guppies were exposed to temephos (10 mg/L) and spinosad (0.5 mg/L) for 7, 14, and 21 days. Afterwards, they were placed in pesticide-free fish tanks (7, 35, and 70 days) for recovery. The results showed that exposure to temephos caused leukocyte death, even at 35 days of recovery. Contrarily, the exposure to spinosad did not cause leukocyte death. This research show, for the first time, that a single dose of temephos causes apoptosis up to 56 days post-exposition, indicating that this pesticide induces chronic effects on immune response cells.

Ex vivo treatment with fucoidan of mononuclear cells from SARS-CoV-2 infected patients.

COVID-19 is a worldwide health emergency, therapy for this disease is based on antiviral drugs and immunomodulators, however, there is no treatment to effectively reduce the COVID-19 mortality rate. Fucoidan is a polysaccharide obtained from marine brown algae, with anti-inflammatory, antiviral, and immune-enhancing properties, thus, fucoidan may be used as an alternative treatment (complementary to prescribed medical therapy) for the recovery of COVID-19.  This work aimed to determine the effects of ex-vivo treatment with fucoidan on cytotoxicity, apoptosis, necrosis, and senescence, besides functional parameters of calcium flux and mitochondrial membrane potential (ΔΨm) on human peripheral blood mononuclear cells isolated from SARS-CoV-2 infected, recovered and healthy subjects. Data suggest that fucoidan does not exert cytotoxicity or senescence, however, it induces the increment of intracellular calcium flux. Additionally, fucoidan promotes recovery of ΔΨm in PBMCs from COVID-19 recovered females. Data suggest that fucoidan could ameliorate the immune response in COVID-19 patients.

Ameloblastomas mimicking apical periodontitis: a case series.

BACKGROUND: Ameloblastomas are benign odontogenic tumors that can eventually mimic the clinical and radiological features of apical periodontitis. The aim of the present study was to evaluate the clinical, radiological and histological characteristics from a series of ameloblastomas mimicking apical periodontitis diagnosed in a 14-year period. MATERIAL AND METHODS: all cases histologically diagnosed as ameloblastomas from 2005 to 2018 presenting a clinical diagnosis of periapical lesion of endodontic origin were selected for the study. Clinical, radiological and histological characteristics from all cases were tabulated and descriptively and comparatively analyzed. RESULTS: Twenty cases composed the final sample, including 18 solid and 2 unicystic ameloblastomas. Mean age of the affected patients was in the fifth decade with predilection for males (72%). The most common anatomical location was the posterior mandible (55%) and most cases presented a radiolucent unilocular (80%) well-defined (95%) image. Most cases were asymptomatic, but the presence of local swelling and bone cortical rupture were common. CONCLUSIONS: Ameloblastomas mimicking periapical lesions of endodontic origin are mostly diagnosed in adult males as well-defined radiolucent unilocular lesions producing local swelling and bone cortical rupture.

Primary total hip arthroplasty with a fully porous-coated uncemented stem: up to twenty-eight years. Retrospective cohort study.

PURPOSE: The use of cementless prosthesis has increased in the last 30 years with the aim of improving the long-term results of total hip arthroplasties in young and active patients. Encouraging results have recently been reported for cementless titanium and cobalt chromium stems. However, there are few studies with long-term follow-up, and the majority have analysed several models of uncemented stems due to their modifications over the years. Therefore, the aim was to assess the long-term survival rate of the Mittelmeier Mark III or Autophor 900-S stem. METHODS: A retrospective cohort study of both gender patients under 70 years old with at least one implanted Mittelmeier Mark III uncemented stem was performed. Survival rate was defined as the proportion of stems that did not need a surgical revision from any cause. Clinical status was evaluated using the Merle d'Aubigne scale modified by Matta (excellent/good/fair/poor). RESULTS: Between 1990 and 1999, 73 stems were implanted. The mean (SD) age at surgical time was 49.3 (9.9) years, and the median (range) of follow-up was 22 (1-28) years. The overall survival rate was 93% (68/73, 95%CI: 85-97%). The stem revisions were due to stem breakage (n = 2), to aseptic loosening (n = 2) and to septic loosening (n = 1). Clinical results were: excellent 84%, good 15% and fair 1.5%. CONCLUSIONS: The Mittelmeier Mark III stem had an excellent survival rate with a stable long-term fixation and excellent clinical outcomes.

Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1ß, -6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

Expression of mBD4, mBD3 and CRAMP during type II collagen-induced arthritis/CIA and their association with inflammation and bone-remodeling markers.

The aim of this study was to analyze the expression of mBD4, mBD3 and CRAMP in joint of mice with type II collagen-induced arthritis/CIA and to explore its possible association with IL-10, IL-4, IFN-γ, IL-17, MMP3, RANK/RANKL/OPG and histological parameters. METHODS: CIA was induced in 44 DBA/1 J mice. The joints from mice were classified into the onset, peak and remission phase of CIA. Histological sections were stained with hematoxylin-eosin and safranin O. The expression of CRAMP, mBD-3, mBD-4, and MMP-3 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression of IL-10, IL-4, IFN-γ, IL-17, RANK/RANKL/OPG was analyzed by RT-PCR. RESULTS: We observed that inflammation and immunostained cells for CRAMP increased in the peak and remission phases compared to the control group. In addition, increments in relative expressions of CRAMP were detected for the remission phase and in IL-4 and IL-17 in the peak phase compared to the control and onset phase. In addition, an increase in IL-10 in a peak phase compared to the control, as well as the relative expression of IFN-γ in remission phase was higher than in the onset phase. This was accompanied by an increase in cartilage damage in the peak phase compared to the control. Cells immunostained to MMP3 increased in the peak phase compared to the onset and control group, and relative expression of MMP3 was detected in the peak phase compared to the onset, remission, and control group. We observed that the relative expression of RANK and RANKL in the peak phase was higher than in control and onset phase. Finally, the relative expression of OPG in the peak phase compared to the onset, remission, and control group was detected. Regarding CRAMP behavior in the different phases studied, it was positively correlated with IL-4 and RANK, and showed a negative correlation with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio in the control group. Also was positively correlated with IFN-γ, IL-17, IL-4, IL-10, as well as with RANK, RANKL, and OPG in the onset and peak phases of the CIA. In the peak phase, CRAMP showed a positive association with MMP3, and we observed a direct correlation between CRAMP and IFN-γ and RANKL/OPG ratio in remission phase. mBD3 correlates positively with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio, and showed a negative correlation with CRAMP, MMP3, and RANK in the control group. Also, it was directly associated with IFN-γ, IL-17, IL-4, IL-10 and RANKL in the onset phase while it was inversely associated with CRAMP, MMP-3, RANK, RANKL, and OPG in the peak phase. Finally, mBD3 was inversely correlated with MMP3 in the remission phase and was directly associated with CRAMP, IFN-γ and RANKL/OPG ratio in this phase. mBD4 was directly associated with CRAMP, IFN-γ, IL-17, IL-4, IL-10, RANKL / OPG in the onset phase, and with CRAMP, IFN-γ, IL-17, IL-4, IL-10, MMP3, RANK, RANKL and OPG in the peak phase. Finally, mBD4 was positively associated with mBD3, IFN-γ, IL-17, IL-10, RANK, RANKL OPG and RANKL/OPG in the CIA remission phase. CONCLUSIONS: Our results demonstrate that CRAMP plays an important role in CIA progress and suggest that its abundance is associated with local pro- and anti-inflammatory status. This makes us propose CRAMP as a possible contributor of bone reconstruction in the last stage of CIA.

Characterization of a non-glycosylated fraction from honey proteins of Melipona beecheii with antimicrobial activity against Escherichia coli O157:H7.

AIMS: To analyse the non-glycosylated protein fraction from Melipona beecheii honey for antimicrobial activity against Escherichia coli O157:H7. METHODS AND RESULTS: The proteins from M. beecheii honey were separated according to their degree of glycosylation using Concanavalin A-affinity chromatography. The total protein extract and its fractions were analysed by 1D and 2D electrophoresis. We also determined the antimicrobial and antihaemolytic activities of the total protein extract and the non-glycosylated fraction. Furthermore, we evaluated the effect of this non-glycosylated fraction for the expression of the Stx1, Stx2, EAE and HlyA pathogen genes. Melipona beecheii honey contained at least 24 proteins with molecular weights ranging between 7·6 and 95 kDa and isoelectric points between 3 and 10, three proteins from the 24 are non-glycosylated. The non-glycosylated fraction had an MIC90 of 1·128 µg ml-1 , and this fraction inhibited the haemolytic activity of the pathogen, as well as reduced the expression of Stx1, Stx2 and HlyA. The MbF1-2 protein from the non-glycosylated fraction was sequenced and identified as a homologue of the royal jelly-like protein of Melipona quadrifasciata. CONCLUSIONS: The non-glycosylated protein fraction from M. beecheii honey greatly contributes to antibacterial activity and it is composed of at least three proteins, of which MbF1-2 provided over 50% of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed significant antimicrobial activity from several proteins present in the honey of M. beecheii. Interestingly, the non-glycosylated protein fraction demonstrated antihaemolytic activity and adversely affected the expression of virulence genes in Escherichia coli O157:H7; these proteins have the potential to be used in developing therapeutic agents against this bacterium.

Quantitative proteomic analysis of marine biofilms formed by filamentous cyanobacterium.

Cyanobacterial molecular biology can identify pathways that affect the adhesion and settlement of biofouling organisms and, consequently, obtain novel antifouling strategies for marine applications. Proteomic analyses can provide an essential understanding of how cyanobacteria adapt to different environmental settings. However, only a few qualitative studies have been performed in some cyanobacterial strains. Considering the limited knowledge about protein expression in cyanobacteria in different growing conditions, a quantitative proteomic analysis by LC-MS/MS of biofilm cells from a filamentous strain was performed. Biofilms were also analysed through standard methodologies for following cyanobacterial biofilm development. Biofilms were formed on glass and perspex at two relevant hydrodynamic conditions for marine environments (average shear rates of 4 s-1 and 40 s-1). Biofilm development was higher at 4 s-1 and no significant differences were found between surfaces. Proteomic analysis identified 546 proteins and 41 were differentially expressed. Differences in protein expression were more noticeable between biofilms formed on glass and perspex at 4 s-1. When comparing biofilms formed on different surfaces, results suggest that biofilm development may be related to the expression of several proteins like a beta-propeller domain-containing protein, chaperone DnaK, SLH domain-containing proteins, an OMF family outer membrane protein, and/or additional uncharacterized proteins. Regarding the hydrodynamic effect, biofilm development can be related to SOD enzyme expression, to proteins related to photosynthetic processes and to a set of uncharacterized proteins with calcium binding domains, disordered proteins, and others involved in electron transfer activity. Studies that combine distinct approaches are essential for finding new targets for antibiofilm agents. The characterisation performed in this work provides new insights into how shear rate and surface affect cyanobacterial biofilm development and how cyanobacteria adapt to these different environmental settings from a macroscopic standpoint to a proteomics context.