New biocatalyst produced from fermented biomass: improvement of adsorptive characteristics and application in aroma synthesis.

This study presents a novel approach to producing activated carbon from agro-industrial residues, specifically cocoa fruit peel, using solid-state fermentation (SSF) with Aspergillus niger. The process effectively degrades lignin, a major impediment in traditional activated carbon production, resulting in a high-quality carbon material. This carbon was successfully utilized for enzyme immobilization and aroma synthesis, showcasing its potential as a versatile biocatalyst. The study meticulously evaluated the physical and chemical attributes of activated carbon derived from fermented cocoa peel, alongside the immobilized enzymes. Employing a suite of analytical techniques-electrophoresis, FTIR, XRD, and TG/DTG the research revealed that fermentation yields a porous material with an expansive surface area of 1107.87 m2/g. This material proves to be an excellent medium for lipase immobilization. The biocatalyst fashioned from the fermented biomass exhibited a notable increase in protein content (13% w/w), hydrolytic activity (15% w/w), and specific activity (29% w/w), underscoring the efficacy of the fermentation process. The significant outcome of this research is the development of a sustainable method for activated carbon production that not only overcomes the limitations posed by lignin but also enhances enzyme immobilization for industrial applications. The study's findings have important implications for the agro-industrial sector, promoting a circular economy and advancing sustainable biotechnological processes.

Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Men's Social Connectedness in Later Life: A Qualitative Study with Older Men.

This qualitative study aimed to understand men's social connectedness in later life in Portugal focusing on their perceptions, obstacles, strategies, and impact on well-being. The sample included 104 older Portuguese men over 65 years of age (Mage = 70.76 years). The qualitative data were the direct transcriptions of the answers given by participants to the electronic interview using thematic analysis. Findings revealed six overarching themes encompassing 18 subcategories: definitions of social connectedness (social support, community identity, mental health promotion, use of community structures), difficulties/obstacles in maintaining social connectedness (ageism, lack of initiative, physical limitations, psychological traits, resources), strategies/actions or resources to establish social connections (use of technology, use of community groups, leisure and sport activities, church/religion), negative impact of difficulties in establishing relevant social connections (mental health, physical health, relationships), positive actions from being socially connected (positive prescriptions to promote social connectedness), and concerns from being socially disconnected (health risks). These findings indicate that the lack of social connectedness creates social vulnerability in later life, and social support is needed to ensure safer aging among older men.

Effect of varying functional monomers in experimental self-adhesive composites: polymerization kinetics, cell metabolism influence and sealing ability.

The aim was to evaluate the effects of adding different functional monomers to experimental self-adhesive composites (SACs) on polymerization kinetics, cell metabolic activity, and sealing ability. SACs were formulated using urethane dimethacrylate as the base monomer and triethylene glycol dimethacrylate. Additionally, 10 wt.% of distinct functional monomers were added - 10-methacryloyloxydecyl dihydrogen phosphate, glycerol phosphate dimethacrylate (GPDM), 2-hydroxyethyl methacrylate (HEMA) or hydroxyethyl acrylamide (HEAA). ATR-FTIR was used to determine real-time polymerization kinetics (20 min,n= 3). The final extrapolated conversion and polymerization rates were determined (DC,max;Rp,max). TheDC,maxvalues were employed to calculate volumetric shrinkage. The MTT assay was performed on MDPC-23 cells using disc extracts at different concentrations (n= 8). Class V cavities were prepared in 60 sound human molars, assigned to six groups (n= 10), depending on the composite used and aging type (T0 or TC, if thermocycled for 10 000 cycles). One-way ANOVA, two-way, andKruskal-Wallistests were employed to treat the data (ɑ= 0.05). Varying the functional monomers had a large impact on DC,max, as confirmed by one-way ANOVA (p<0.001). The highest was obtained for HEMA (64 ± 3%). The HEMA and HEAA formulations were found to be significantly more toxic at concentrations below 100%. For microleakage, having a functional monomer or not did not show any improvement, irrespective of margin or aging period (Mann-Whitney U,p> 0.05). Larger functional monomers MDP and GPDM affected polymerization properties. Conversely, their acidity did not seem to be detrimental to cell metabolic activity. Regarding sealing ability, it seems that the functional monomers did not bring an advantage to the composites. Varying the functional monomer in SACs had a clear impact on the polymerization kinetics as well as on their cytotoxic potential. However, it did not confer better microleakage and sealing. Claiming self-adhesiveness based only on functional monomers seems dubious.

Synthesis of hexyl butyrate (apple and citrus aroma) by Candida rugosa lipase immobilized on Diaion HP-20 using the Box-Behnken design.

This study aims at the synthesis of hexyl butyrate by Candida rugosa lipase (CRL) immobilized on Diaion HP 20. The lipase load used was 28.7 ± 2.1 mg/g (mg of lipase/g of support), whose hydrolytic activity was 132.0 ± 2.5 U/g. To obtain the maximum production of hexyl butyrate, the Box-Behnken design statistical planning was used, having as independent variables; biocatalyst concentration, temperature and acid:alcohol molar ratio and ester conversion as a dependent variable at 60, 180 and 480 min. For 60 min, 90.8% conversion was obtained at 47.25 ºC, 1:1.4 molar ratio and 17.65% of biocatalyst; 180 min, 94.5% conversion at 59.5 ºC, 1:2 molar ratio and 15.8% biocatalyst; 480 min, 95.01% conversion at 47.0 ºC, 1:2 molar ratio and 16.9% biocatalyst. CRL-Diaion HP 20 retained 60% of its initial activity after ten cycles of reactions showing potential for industrial use. The ester produced was identified by gas chromatography analyses. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01200-1.

Rare Case of Rickettsiosis Caused by Rickettsia monacensis, Portugal, 2021.

We report a case of rickettsiosis caused by Rickettsia monacensis in an immunocompetent 67-year-old man in Portugal who had eschar, erythematous rash, and an attached Ixodes ricinus tick. Seroconversion and eschar biopsy led to confirmed diagnosis by PCR. Physicians should be aware of this rare rickettsiosis, especially in geographic regions with the vector.

Impact of Maternal Feed Restriction at Different Stages of Gestation on the Proteomic Profile of the Newborn Skeletal Muscle.

We aimed to investigate the effects of the maternal plane of nutrition during gestation on the proteome profile of the skeletal muscle of the newborn. Pregnant goats were assigned to the following experimental treatments: restriction maintenance (RM) where pregnant dams were fed at 50% of their maintenance requirements from 8−84 days of gestation, and then feed of 100% of the maintenance requirements was supplied from 85­parturition (n = 6); maintenance restriction (MR) where pregnant dams were fed at 100% of their maintenance requirements from 8−84 days of gestation, and then experienced feed restriction of 50% of the maintenance requirements from 85­parturition (n = 8). At birth, newborns were euthanized and samples of the Longissimus dorsi muscle were collected and used to perform HPLC-MS/MS analysis. The network analyses were performed to identify the biological processes and KEGG pathways of the proteins identified as differentially abundant protein and were deemed significant when the adjusted p-value (FDR) < 0.05. Our results suggest that treatment RM affects the energy metabolism of newborns' skeletal muscle by changing the energy-investment phase of glycolysis, in addition to utilizing glycogen as a carbon source. Moreover, the RM plane of nutrition may contribute to fatty acid oxidation and increases in the cytosolic α-KG and mitochondrial NADH levels in the skeletal muscle of the newborn. On the other hand, treatment MR likely affects the energy-generation phase of glycolysis, contributing to the accumulation of mitochondrial α-KG and the biosynthesis of glutamine.

Antiseptic barrier caps in central line-associated bloodstream infections: A systematic review and meta-analysis.

OBJECTIVES: To evaluate the evidence concerning the effectiveness of antiseptic barrier caps vs. manual disinfection in preventing central line-associated bloodstream infection (CLABSI). METHODS: The protocol of this systematic review and meta-analysis was pre-registered in PROSPERO (CRD42021259582). PubMed, Cochrane Library and Web of Science databases were searched from 2011 to 2021. Randomized-controlled trials (RCT) and observational studies on hospitalized patients of any age were included. RESULTS: Fourteen studies were included. Compared with manual disinfection, antiseptic barrier caps significantly reduced CLABSI rate per 1000 line-days (Standardized Mean Difference [SMD]: -0.02; 95%CI: -0.03 to -0.01) and number of CLABSI per patient (RR: 0.60; 95%CI: 0.41-0.89). Subgroup analysis showed that antiseptic barrier caps were more effective in reducing CLABSI rate per 1000 line-days in ICU (SMD: -0.02; 95%CI: -0.03 to -0.01) and non-ICU patients (SMD: -0.03; 95%CI: -0.05 to -0.01), adults (SMD: -0.02; 95%CI: -0.04 to -0.01), as in observational studies (SMD: -0.02; 95%CI: -0.02 to -0.01). Antiseptic barrier caps also significantly reduce CLABSI risk in ICU patients (RR: 0.65, 95%CI: 0.42-1.00), adults (RR: 0.50, 95%CI: 0.29-0.86), and observational studies (RR: 0.54; 95%CI: 0.32-0.91). No differences were found when only children or RCTs were taken into account. Median cost savings amongst studies were $21,890 [IQR 16,350-45,000] per CLABSI. CONCLUSIONS: Antiseptic barrier caps appear to be effective in reducing CLABSI. The real-world impact needs to be confirmed by RCTs.

Candida rugosa lipase immobilized on hydrophobic support Accurel MP 1000 in the synthesis of emollient esters.

OBJECTIVES: To immobilize Candida rugosa lipase in Accurel MP 1000 (CRL-AMP) by physical adsorption in organic medium and apply in the synthesis of wax esters dodecanoyl octadecanoate 1 and hexadecanoyl octadecanoate 2 in a heptane medium, as well as evaluating the stability and recyclability of CRL-AMP in six reaction cycles. RESULTS: The specific activity (Asp) for CRL-AMP was 200 ± 20 U mg-1. Its catalytic activity was 1300 ± 100 U g-1. CRL-AMP was used in the synthesis of esters in heptane medium with a 1:1 acid:alcohol molar ratio at 45 °C and 200 rpm. In synthesis 1, conversion was 62.5 ± 3.9% in 30 min at 10% m v-1 and 56.9 ± 2.8% in 54 min at 5% m v-1; while in synthesis 2, conversion was 79.0 ± 3.9% in 24 min at 10% m v-1, and 46.0 ± 2.4% in 54 min at 5% m v-1. Reuse tests after six consecutive cycles of reaction showed that the biocatalyst retained approximately 50% of its original activity for both reaction systems. CONCLUSIONS: CRL-AMP showed a high potential in the production of wax esters, since it started from low enzymatic load and high specific activities and conversions were obtained, in addition to allowing an increase in stability and recyclability of the prepared biocatalyst.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification.

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain-Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

A Thorn in a Haystack: A Rare Case of Septic Arthritis.

Pantoea agglomerans is a facultative anaerobe and environmental bacterium that could be a cause of opportunistic human infections, especially in wound infection with plant material. Arthritis or synovitis secondary to penetrating plant thorn injuries is not frequently reported. We present the case of a 35-year-old otherwise healthy male with a bramble thorn penetrating injury of the left knee. P. agglomerans was isolated from the synovial fluid. The patient was treated with amoxicillin/clavulanate according to sensitivity testing. This case highlights the importance of precise and thorough medical history, especially for less common presentations, as well as source control.

Application of lipase immobilized on a hydrophobic support for the synthesis of aromatic esters.

The present study aimed at preparing three biocatalysts via physical adsorption of lipases from Candida rugosa (CRL), Mucor javanicus, and Candida sp. on a hydrophobic and mesoporous support (Diaion HP-20). These biocatalysts were later applied to the synthesis of aromatic esters of apple peel and citrus (hexyl butyrate), apple and rose (geranyl butyrate), and apricot and pineapple (propyl butyrate). Scanning electron microscopy and gel electrophoresis confirmed a selective adsorption of lipases on Diaion, thus endorsing simultaneous immobilization and purification. Gibbs free energy (∆G) evinced the spontaneity of the process (-17.9 kJ/mol ≤ ∆G ≤ -5.1 kJ/mol). Maximum immobilized protein concentration of 30 mg/g support by CRL. This biocatalyst was the most active in olive oil hydrolysis (hydrolytic activity of 126.0 ± 2.0 U/g) and in the synthesis of aromatic esters. Maximum conversion yield of 89.1% was attained after 150 Min for the synthesis of hexyl butyrate, followed by the synthesis of geranyl butyrate (87.3% after 240 Min) and propyl butyrate (80.0% after 150 Min). CRL immobilized on Diaion retained around 93% of its original activity after six consecutive cycles of 150 Min for the synthesis of hexyl butyrate.

A Rare Form of Metastatic Melanoma in an HIV-Infected Patient - A Diagnosis to Remember.

Malignant melanoma (MM), which is amongst the rarest skin cancers, still remains one of the deadliest and most likely to spread, and, in human immunodeficiency virus (HIV)-infected patients, generally has a more aggressive behaviour. Although gastrointestinal (GI) tract metastases are frequent, secondary symptomatic colonic disease is rare. We present the case of a 76-year-old HIV-infected patient, with a 15-month history of GI and constitutional symptoms and a previous diagnosis of malignant melanoma. Diagnostic workup revealed metastatic involvement of the cecum. This case highlights the need to bear in mind the metastatic involvement of the GI tract by MM, and MM itself, especially in HIV-infected patients.

TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT.

Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells.

Heteroduplex mobility assay and single-stranded conformation polymorphism analysis as methodologies for detecting variants of human erythroviruses.

Variant samples from the three genotypes of erythroviruses have already been detected using sequencing as methodology for analysis. This study aimed to investigate the efficacy of single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assay (HMA) as methodologies to detect human erythrovirus variants, using their VP1 unique region sequences. Clinical samples and plasmids of PVBAUA, A6, LaLi, V9Gh3051, and D91.1 erythrovirus variants as prototypes of the three genotypes were used. SSCP analysis was able to distinguish all divergences among the plasmids, including the two mutation points between LaLi and A6 plasmids that led to distinct electrophoresis mobility patterns. Although HMA analysis was unabled to detect two mutation points between LaLi and A6, it enabled the differentiation among all other plasmids that revealed specific electrophoresis patterns, with high-enough sensibility to detect 1.5% nucleotide substitutions. When 57 clinical samples were analyzed, 33 of them presented an identical pattern to PVBAUA by HMA and SSCP analyses, two of them were sequenced and presented an identical sequence in relation to PVBAUA. Another pattern was found for 21 samples. Among these, two samples were sequenced, revealing one mutation point in relation to PVBAUA, while each one of the three remaining samples presented a distinct pattern, showing two or three mutations in relation to PVBAUA by sequencing. HMA and SSCP analyses were suggested as methodologies suited for detecting genetic mutations of human erythroviruses in developing countries because of their practicability and minor costs for reagents and equipment.

MIB-1 labelling indices according to clinico-pathological variables in canine mammary tumours: a multivariate study.

The relationship between MIB-1 labelling indices (LI), as detected by immunohistochemical methods, and other clinico-pathological characteristics was studied in a series of 77 malignant mammary tumours surgically removed from 47 female dogs. The immunostaining was assessed on the basis of the estimated percentage of positive cells in the areas of highest labelling. Multivariate logistic regression demonstrated no influence of breed, age, previous pregnancies, previous progestin administration, histological type or location of the tumour on MIB-1 LI. MIB-1 LI was significantly related to the size of the tumour, necrosis, invasive growth and histological grade, but not with ulceration, lymph node metastasis, skin fixation or E-cadherin expression. The significant relationship between MIB-1 LI and other known factors of poor prognosis suggests that a high LI may have prognostic value in canine malignant mammary tumours.

A novel monoclonal antibody against canine monocytes/macrophages.

The production and partial characterization of a monoclonal antibody, the IgG1 IH1, which recognizes an antigen distributed in canine monocytes/macrophages, is reported here. The distribution and apparent molecular weight of the antigen recognized by the IH1 MAb was determined in peripheral blood leukocytes, peripheral blood monocyte-derived macrophages and tissue sections of spleen, liver and skin, using Western blotting, immunocytochemistry, immunohistochemistry and flow cytometry. The IH1 MAb-recognized antigen was detected in Western blotting under non-reducing conditions spread out as a large band covering the position corresponding to the migration of molecules with molecular weights from 55 to 73 kDa. The IH1 MAb labeled blood monocytes, tissue macrophages in lymph nodes, and in the mantle zone of the spleen, and Kupffer cells in the liver. It did not react with human cells. In flow cytometric analysis, the IH1 MAb reacted with a subpopulation of monocytes. The MAb described herein may become a valuable tool for diagnosis and research on canine diseases.