Unraveling resistance mechanisms in combination therapy: A comprehensive review of recent advances and future directions.

Antimicrobial resistance is a global health threat. Misuse and overuse of antimicrobials are the main drivers in developing drug-resistant bacteria. The emergence of the rapid global spread of multi-resistant bacteria requires urgent multisectoral action to generate novel treatment alternatives. Combination therapy offers the potential to exploit synergistic effects for enhanced antibacterial efficacy of drugs. Understanding the complex dynamics and kinetics of drug interactions in combination therapy is crucial. Therefore, this review outlines the current advances in antibiotic resistance's evolutionary and genetic dynamics in combination therapies-exposed bacteria. Moreover, we also discussed four pivotal future research areas to comprehend better the development of antibiotic resistance in bacteria treated with combination strategies.

Synthesis and Design of a Synthetic-Living Material Composed of Chitosan, Calendula officinalis Hydroalcoholic Extract, and Yeast with Applications as a Biocatalyst.

Design and development of materials that couple synthetic and living components allow taking advantage of the complexity of biological systems within a controlled environment. However, their design and fabrication represent a challenge for material scientists since it is necessary to synthesize synthetic materials with highly specialized biocompatible and physicochemical properties. The design of synthetic-living materials (vita materials) requires materials capable of hosting cell ingrowth and maintaining cell viability for extended periods. Vita materials offer various advantages, from simplifying product purification steps to controlling cell metabolic activity and improving the resistance of biological systems to external stress factors, translating into reducing bioprocess costs and diversifying their industrial applications. Here, chitosan sponges, functionalized with Calendula officinalis hydroalcoholic extract, were synthesized using the freeze-drying method; they showed small pore sizes (7.58 µm), high porosity (97.95%), high water absorption (1695%), and thermal stability, which allows the material to withstand sterilization conditions. The sponges allowed integration of 58.34% of viable Saccharomyces cerevisiae cells, and the cell viability was conserved 12 h post-process (57.14%) under storage conditions [refrigerating temperature (4 °C) and without a nutrient supply]. In addition, the synthesized vita materials conserved their biocatalytic activity after 7 days of the integration process, which was evaluated through glucose consumption and ethanol production. The results in this paper describe the synthesis of complex vita materials and demonstrate that biochemically modified chitosan sponges can be used as a platform material to host living and metabolically active yeast with diverse applications as biocatalysts.

Long-Term Performance of a Hybrid-Flow Constructed Wetlands System for Urban Wastewater Treatment in Caldera de Tirajana (Santa Lucía, Gran Canaria, Spain).

This paper describes the results that have been obtained in a real case study of a hybrid constructed wetlands system, which has been in continuous operation for over 11 years. The main aim of the study was to understand the long-term operation and efficiency of the system (which is situated in the municipality of Santa Lucía, Gran Canaria, Spain), which comprises two vertical-flow and one horizontal-flow constructed wetlands for the treatment of urban wastewater. The system, which was originally designed to treat a flow rate of 12.5 m3/day, with a load of 100 equivalent inhabitants, has been operating since its inauguration (July 2008), with a flow rate of almost 35 m3/day and a load of 400 equivalent inhabitants. Despite this, the mean total removal efficiencies during the study period (2014-2019) are optimal for a system of these characteristics, as follows: 92% for 5-day biochemical oxygen demand (BOD5), 89% for the chemical oxygen demand (COD), and 97% for the total suspended solids (TSS). The system efficiency, with respect to nutrient removal, was somewhat lower, resulting in 48% for total N and 35% for NH4. It has been confirmed with this study that this type of system is an appropriate, robust, resilient nature-based solution for the treatment of the wastewater that is generated in small communities, especially in zones with a warm climate, stable mean temperatures, and mild winters.

Susceptibility for Some Infectious Diseases in Patients With Diabetes: The Key Role of Glycemia.

Uncontrolled diabetes results in several metabolic alterations including hyperglycemia. Indeed, several preclinical and clinical studies have suggested that this condition may induce susceptibility and the development of more aggressive infectious diseases, especially those caused by some bacteria (including Chlamydophila pneumoniae, Haemophilus influenzae, and Streptococcus pneumoniae, among others) and viruses [such as coronavirus 2 (CoV2), Influenza A virus, Hepatitis B, etc.]. Although the precise mechanisms that link glycemia to the exacerbated infections remain elusive, hyperglycemia is known to induce a wide array of changes in the immune system activity, including alterations in: (i) the microenvironment of immune cells (e.g., pH, blood viscosity and other biochemical parameters); (ii) the supply of energy to infectious bacteria; (iii) the inflammatory response; and (iv) oxidative stress as a result of bacterial proliferative metabolism. Consistent with this evidence, some bacterial infections are typical (and/or have a worse prognosis) in patients with hypercaloric diets and a stressful lifestyle (conditions that promote hyperglycemic episodes). On this basis, the present review is particularly focused on: (i) the role of diabetes in the development of some bacterial and viral infections by analyzing preclinical and clinical findings; (ii) discussing the possible mechanisms by which hyperglycemia may increase the susceptibility for developing infections; and (iii) further understanding the impact of hyperglycemia on the immune system.

Enhanced production of taxadiene in Saccharomyces cerevisiae.

BACKGROUND: Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. RESULTS: Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. CONCLUSIONS: The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.

The Demand for New Antibiotics: Antimicrobial Peptides, Nanoparticles, and Combinatorial Therapies as Future Strategies in Antibacterial Agent Design.

The inappropriate use of antibiotics and an inadequate control of infections have led to the emergence of resistant strains which represent a major threat to public health and the global economy. Therefore, research and development of a new generation of antimicrobials to mitigate the spread of antibiotic resistance has become imperative. Current research and technology developments have promoted the improvement of antimicrobial agents that can selectively interact with a target site (e.g., a gene or a cellular process) or a specific pathogen. Antimicrobial peptides and metal nanoparticles exemplify a novel approach to treat infectious diseases. Nonetheless, combinatorial treatments have been recently considered as an excellent platform to design and develop the next generation of antibacterial agents. The combination of different drugs offers many advantages over their use as individual chemical moieties; these include a reduction in dosage of the individual drugs, fewer side effects compared to the monotherapy, reduced risk for the development of drug resistance, a better combined response compared to the effect of the individual drugs (synergistic effects), wide-spectrum antibacterial action, and the ability to attack simultaneously multiple target sites, in many occasions leading to an increased antibacterial effect. The selection of the appropriate combinatorial treatment is critical for the successful treatment of infections. Therefore, the design of combinatorial treatments provides a pathway to develop antimicrobial therapeutics with broad-spectrum antibacterial action, bactericidal instead of bacteriostatic mechanisms of action, and better efficacy against multidrug-resistant bacteria.

Metabolic Engineering and Synthetic Biology: Synergies, Future, and Challenges.

The "-omics" era has brought a new set of tools and methods that have created a significant impact on the development of Metabolic Engineering and Synthetic Biology. These fields, rather than working separately, depend on each other to prosper and achieve their individual goals. Synthetic Biology aims to design libraries of genetic components (promoters, coding sequences, terminators, transcriptional factors and their binding sequences, and more), the assembly of devices, genetic circuits and even organism; in addition to obtaining quantitative information for the creation of models that can predict the behavior of biological systems (Cameron et al., 2014). Metabolic engineering seeks for the optimization of cellular processes, endemic to a specific organism, to produce a compound of interest from a substrate, preferably cheap and simple. It uses different databases, libraries of components and conditions to generate the maximum production rate of a desired chemical compound and avoiding inhibitors and conditions that affect the growth rate and other vital functions in the specific organism to achieve these goals; metabolic fluxes manipulation represents an important alternative (Stephanopoulos, 2012).

Hybrid single-port cholecystectomy in children.

BACKGROUND AND OBJECTIVES: Multiple single-port or single-incision techniques have been successfully implemented for laparoscopic cholecystectomy in adults and children. These techniques require either a large multichannel port or a larger skin incision to accommodate multiple ports or instruments. Inspired by a first generation single-port instrument, we developed a safe and effective technique for a single-port laparoscopic cholecystectomy with virtually scarless results. METHODS: Over a 14-mo period, 20 patients (19 females, 1 male) underwent the hybrid single-port cholecystectomy. A straight 10-mm Storz telescope with inbuilt 6-mm working channel in combination with 2 portless 2.3-mm percutaneous graspers was used. The dissection is carried out with 43-cm bariatric length instruments. The cystic artery and duct are sealed with WECK Hem-o-lok clips or the Harmonic scalpel. RESULTS: Range (mean) age: 7.7 y to 19.5 y (15.5), BMI: 11.6kg/m(2) to 42.3kg/m(2) (27), operative duration 48 min to 120 min (79), postoperative length of stay: 5 h to 78 h (24). DIAGNOSIS: 13 patients cholecystolithiasis, 7 patients biliary dyskinesia. Conversion to conventional 4-port cholecystectomy was required in 2 patients. No intra- or postoperative complications occurred. CONCLUSION: The hybrid single-port technique is easy to master. It provides traditional anatomical exposure and allows application of conventional laparoscopic principles.

Heat shock proteins (HSP70 and HSP27) as markers of epithelial dysplasia in oral leukoplakia.

Heat shock proteins (HSPs) play a significant role in cell proliferation, differentiation, and oncogenesis. HSP70 and HSP27 are constitutively and gradually expressed in a broad range of normal tissues and neoplasms, and their expression has been assessed as markers for oral epithelial dysplasia. The study involved 43 patients with oral leukoplakia (OL): 23 were categorized as nondysplastic and 20 as dysplastic OLs. Immunohistochemistry was carried out with the monoclonal antibodies HSP70 and HSP27. The presence of epithelial dysplasia and its histologic grading was evaluated according to the World Health Organization classification: mild, moderate, and severe squamous epithelial dysplasia. Expression of HSPs within the epithelium was also evaluated. The difference in the percentage of HSP70 positive nuclei in nondysplastic and dysplastic OL reached statistical significance(Equation is included in full-text article.)95% confidence interval = 17.74-43.82; P = 0.000). None of the 43 specimens analyzed showed positive nuclear immunostaining for anti-HSP27 antibody. No significant difference for HSP27 cytoplasmic expression could be identified between OL with or without epithelial dysplasia(Equation is included in full-text article.)95% confidence interval = 0.44-3.95; P = 0.89). It is concluded that the nuclear HSP70 immunoexpression could be an objective marker for the presence of the epithelial dysplasia in OL.

Manejo quirúrgico de enfermedades proctológicas en consulta externa conveniencia económica / Surgical management of proctologic diseases in outpatient consultation economic advantage

Se realizó un estudio sobre el manejo quirúrgico de enfermedades proctológicas en consulta externa del hospital Universitario Camilo Cienfuegos con el objetivo de enumerar las intervenciones realizadas, presentar las complicaciones mas frecuentes en cuanto al manejo quirúrgico y conocer el ahorro económico y la repercusión social en 2000 pacientes operados en consulta externa. Resultados: la hemorroidectomia y la fisurectomia fueron las técnicas mas realizadas con un 40 por ciento y 31.3 por ciento respectivamente, solo se presentaron complicaciones en un 3 por ciento de nuestros enfermos. El manejo quirúrgico de estas enfermedades en consulta externa tiene un significativo impacto económico pues en 2000 pacientes operados se ahorró al hospital por concepto de días/pacientes 167 700 pesos y por concepto de día cama 152.760. Conclusiones: el manejo quirúrgico en consulta de estas enfermedades tiene una importante repercusión económica y una apreciable aceptación social pues el 98 por ciento de los enfermos estuvo de acuerdo con esta modalidad(AU)

A study was made on the surgical handling of proctological diseases in external consultation of the Camilo Cienfuegos University Hospital with the objective of enumerating the interventions carried out, to present the most frequent complications as to the surgical handling and to know the economic saving and the social repercussion in 2000 patients operated in external consultation. Results: haemorrhoidectomy and fissurectomy were the techniques most carried out with a 40 percent and a 31.3 percent, respectively. Complications were present in only a 3 percent of our patients persons. The surgical handling of these illnesses in external consultation has a significant economic impact because in 2000 operated patients you saving to the hospital for concept of días/pacientes 167 700 pesos and for concept by day bed 152.760. Conclusions: the surgical handling in consultation of these illnesses has an important economic repercussion and an appreciable social acceptance because 98% of the sick persons agreed with this modality(AU)

Sangramiento digestivo bajo: estudio de quinquenio / Lower digestive tract bleeding: a five-year study

Se realizó un estudio retrospectivo en el período comprendido desde Enero de 2000 a Diciembre del 2005, acerca de los pacientes que ingresaron con el diagnóstico de Sangramiento digestivo bajo, en el Hospital Universitario Camilo Cienfuegos de Sancti Spíritus, con el objetivo de conocer, edad, sexo, causas del sangramiento, el medio diagnóstico utilizado, necesidad de transfusiones y estadía. Se revisaron 64 expedientes clínicos y su concluyó que los grupos más afectados fue la octava década de la vida, predominó el sexo masculino, el mayor número de pacientes se fue sin diagnóstico, pero como causa más frecuente predominó la diverticulosis del colón, el medio diagnóstico más usado fue la rectosigmoidoscopia, el mayor número de paciente no necesitó transfusiones, la estadía hospitalaria fue de cinco días y el tratamiento quirúrgico no se tuvo en cuenta en estos pacientes(AU)

A retrospective study was carried out in the period from January of 2000 to December of 2005, about the patients admitted with a diagnosis of lower digestive bleeding, at the Camilo Cienfuegos University Hospital of Sancti Spíritus, with the objective of knowing, age, sex, causes of bleeding , diagnostic means used, necessity of transfusions and hospital stay. 64 clinical files were reviewed and it was concluded that the affected groups were the eighth decade of life, the masculine sex prevailed, the greatest number of patients were discharged without a diagnosis, but colon diverticulosis prevailed as the most frequent cause, the most used diagnostic means was the rectosigmoidoscopy, the majority of patients didn't need transfusions, the hospital stay was of five days and the surgical treatment was not taken into account in these patients(AU)

Mutation study of Spanish patients with hereditary hemorrhagic telangiectasia and expression analysis of Endoglin and ALK1.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder originated by mutations in Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. The first large series HHT analysis in Spanish population has identified mutations in 17 unrelated families. Ten different mutations in ALK1 and six in ENG genes were found. Six unrelated families had a mutation in ENG gene, four representing new mutations, p.Y258fs, pV323fs, p.F279fs (c.834_837del CTTC), and p.F279fsdupC. Eleven unrelated families harboured mutations in ALK1; ten were new mutations identified as p.H328P, p.R145fs, p.G68C, p.A377T, p.H297R, p.M376T, p.C36Y, p.H328P, p.T82del and p.R47P. Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1), in agreement with data reported for other Mediterranean countries (France, Italy), but at variance with Northern Europe or North America. Endoglin expression in HHT1 or HHT2 activated monocytes and blood outgrowth endothelial cells (BOECs) from older patients was well below the theoretical 50% level expected from the HHT1 haploinsufficiency model, suggesting that the pathogenic endoglin haploinsufficiency leading to the HHT phenotype is age-dependent. Interestingly, ALK1 protein levels of HHT BOECs in some missense ALK1 mutants were similar to controls. In vitro expression of these ALK1 constructs suggests that, in addition to the haploinsufficiency model, certain ALK1 mutants may inhibit the function of the wild type allele.

Mutation analysis in Spanish patients with hereditary hemorrhagic telangiectasia: deficient endoglin up-regulation in activated monocytes.

BACKGROUND: Mutations in the endoglin (ENG) or ALK1 genes are responsible for hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2), respectively, a dominant vascular dysplasia caused by haploinsufficiency. No formal mutation studies of patients with HHT have been conducted in Spain. METHODS: ENG and ALK1 mutation analyses were carried out in 13 Spanish HHT patients diagnosed according to the Curacao criteria. Because endoglin is up-regulated at the cell surface during the monocyte-macrophage transition, endoglin concentrations in activated monocytes were determined by immunofluorescence flow cytometry in a systematic analysis. As controls, 40 non-HHT volunteers were studied for up-regulation of endoglin in activated monocytes. RESULTS: The mutation responsible for HHT was identified in eight patients belonging to two unrelated families. One of the families has a nonsense mutation in exon 4 (c.511C>T; R171X) of the ENG gene, and accordingly the disorder was identified as HHT1. The other family has a missense mutation affecting exon 8 (c.1120C>T; R374W) of the ALK1 gene, and hence is a HHT2 family. Interestingly, endoglin up-regulation was deficient in activated monocytes of both HHT1 and HHT2 patients compared with controls. By contrast, endoglin up-regulation was age-independent in control donors across a broad range of ages. The extent of endoglin up-regulation in activated monocytes was most diminished in those patients with the most severe symptoms. CONCLUSIONS: Endoglin up-regulation in activated monocytes is impaired in HHT1 and HHT2 patients and is age-dependent in both HHT types. Endoglin expression may predict the clinical severity of HHT.

Lumican is down-regulated in cells expressing endoglin. Evidence for an inverse correlationship between Endoglin and Lumican expression.

Endoglin (CD105) is a homodimeric membrane glycoprotein, which acts as a TGF-beta coreceptor in the vasculature and plays an important role in cardiovascular development and vascular remodelling. To isolate putative genes regulated by endoglin expression, a PCR-based RNA fingerprinting technique was carried out. Myoblasts stably transfected with endoglin showed a decrease in the expression of lumican both at the RNA and protein levels. Lumican is a proteoglycan of the extracellular matrix, belonging to the SLRP (Small Leucine-Rich Repeat Proteoglycans) family. Lumican down-regulation by endoglin appeared to be controlled, at least in part, at the transcriptional level, as indicated by RT-PCR, and transient transfection experiments using a lumican promoter reporter based vector. This inverse correlation between endoglin and lumican expression was substantiated by immunohistochemical staining of vessels from human tissues. Thus, cells belonging to the high endothelia, such as tonsil, express a large amount of endoglin, and the lumican content of their matrix is considerably reduced. Conversely, in resting endothelia, such as that of large vessels, the expression of endoglin is reduced whereas the amount of lumican is greatly increased. The inverse regulation in the expression of endoglin and lumican was also evident after TGF-beta treatments since endoglin was up-regulated, whereas lumican was down-regulated by this cytokine. This report describes for the first time a relationship between endoglin and lumican expression.

A cross-talk between hypoxia and TGF-beta orchestrates erythropoietin gene regulation through SP1 and Smads.

Erythropoietin (Epo) is the humoral regulator of red blood-cell production. Low oxygen tension increases the Epo levels by enhancing transcription, through the hypoxia-inducible factor (HIF)-1, a transcriptional modulator in oxygen-regulated gene expression. In the present work, a cooperative interaction between hypoxia, mediated by the HIF-1 complex, and transforming growth factor-beta (TGF-beta), mediated by Smad3/4, was revealed in the Epo gene. This cooperation is due to physical interaction between Smad3/4 and HIF-1alpha. The Smad3/4 binding site is located within the 3' Epo enhancer, downstream from the HRE consensus, and immediately adjacent to the orphan hepatic nuclear factor receptor (HNF-4). HNF-4 is interacting also with Smad3 and the HIF-1 complex, to potentiate further the cooperative effect between both factors. Moreover, Sp1 has been identified as the factor binding the promoter necessary for the full hypoxia inducibility of the EPO gene. However, this full induction is achieved only if the TGF-beta pathway is mediating a cross-talk between promoter (Sp1) and enhancer (HIF-1alpha) regions through Smad3. We show that Sp1 binding to the proximal promoter is relevant for Epo transcription, and contributes to the Epo induction by hypoxia. A functional cooperation among the transcription factors mediating hypoxia (HIF-1, Sp1), the TGF-beta pathway (Smad3/4), and tissue-specific HNF-4 is proposed for the regulation of the Epo gene. In this model, the physical contact between the upstream promoter and the 3' downstream enhancer is mediated by Sp1 and Smad3 factors, and would occur upon bending of the DNA intervening sequences. Thus, Sp1 would reinforce the promoter/enhancer contact, while Smad3 would stabilize the multifactorial complex by interacting with HIF-1/Sp1/HNF-4 and the coactivator CBP/p300. This model may be extended to other genes where collaboration between TGF-beta and hypoxia takes place.

Transcriptional activation of endoglin and transforming growth factor-beta signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury.

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-beta (TGF-beta) signaling receptors to bind TGF-beta family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-beta, TGF-beta signaling receptors, and TGF-beta-stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglin promoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated the endoglin promoter and those of collagen alpha1(I), urokinase-type plasminogen activator, TGF-beta1, and TGF-beta receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-beta signaling complex in vascular repair.

Molecular and antigenic characterization of the Leishmania (Viannia) panamensis kinetoplastid membrane protein-11

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire coding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was cloned, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97 per cent and 96 per cent respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. The L. (L) panamensis ORF was subsequently cloned in a high expression vector and the recombinant protein was included and purified from Escherichia coli cultures. Immunoblot analysis showed that 80 per cent, 77 per cent and 100 per cent sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86 per cent of a asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We propose that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.