Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Osteopontin Is an Integral Mediator of Cardiac Interstitial Fibrosis in Models of Human Immunodeficiency Virus Infection.

BACKGROUND: People with human immunodeficiency virus (HIV) have heightened incidence/risk of diastolic dysfunction and heart failure. Women with HIV have elevated cardiac fibrosis, and plasma osteopontin (Opn) is correlated to cardiac pathology. Therefore, this study provides mechanistic insight into the relationship between osteopontin and cardiac fibrosis during HIV infection. METHODS: Mouse embryonic fibroblasts (MEFs) modeled cardiac fibroblasts in vitro. Simian immunodeficiency virus (SIV)-infected macaques with or without antiretroviral therapy and HIV-infected humanized mice modeled HIV-associated cardiac fibrosis. RESULTS: Lipopolysaccharide-stimulated MEFs were myofibroblast-like, secreted cytokines, and produced Opn transcripts. SIV-infected animals had elevated plasma Opn at necropsy, full-length Opn in the ventricle, and ventricular interstitial fibrosis. Regression modeling identified growth differentiation factor 15, CD14+CD16+ monocytes, and CD163 expression on CD14+CD16+ monocytes as independent predictors of plasma Opn during SIV infection. HIV-infected humanized mice showed increased interstitial fibrosis compared to uninfected/untreated animals, and systemic inhibition of osteopontin by RNA aptamer reduced left ventricle fibrosis in HIV-infected humanized mice. CONCLUSIONS: Since Opn is elevated in the plasma and left ventricle during SIV infection and systemic inhibition of Opn reduced cardiac fibrosis in HIV-infected mice, Opn may be a potential target for adjunctive therapies to reduce cardiac fibrosis in people with HIV.

Research on Rare Diseases in Germany - The cancer predisposition syndrome registry.

Background: Cancer predisposition syndromes (CPS) are rare diseases that are associated with an increased risk of cancer due to genetic alterations. At least 8 % of all cases of childhood cancer are attributable to CPS [1, 2]. The CPS registry was launched in 2017 to learn more about CPS and to improve the care to those afflicted by these diseases. Methods: This is an internationally networked registry with associated accompanying studies that investigate cancer risks and spectra, the possibilities of cancer prevention, early detection and therapy. Results: For several of these syndromes, new insights into the cancer risks and cancer types as well as factors modifying cancer risk have been gained. In addition, experimental, psycho-oncological, preclinical and clinical studies were initiated. Conclusions: The CPS registry is an example of how progress can be made within a short period of time to the benefit of individuals with rare diseases through systematic data collection and research.

Genotype-phenotype associations within the Li-Fraumeni spectrum: a report from the German Registry.

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by pathogenic TP53 variants. The condition represents one of the most relevant genetic causes of cancer in children and adults due to its frequency and high cancer risk. The term Li-Fraumeni spectrum reflects the evolving phenotypic variability of the condition. Within this spectrum, patients who meet specific LFS criteria are diagnosed with LFS, while patients who do not meet these criteria are diagnosed with attenuated LFS. To explore genotype-phenotype correlations we analyzed 141 individuals from 94 families with pathogenic TP53 variants registered in the German Cancer Predisposition Syndrome Registry. Twenty-one (22%) families had attenuated LFS and 73 (78%) families met the criteria of LFS. NULL variants occurred in 32 (44%) families with LFS and in two (9.5%) families with attenuated LFS (P value < 0.01). Kato partially functional variants were present in 10 out of 53 (19%) families without childhood cancer except adrenocortical carcinoma (ACC) versus 0 out of 41 families with childhood cancer other than ACC alone (P value < 0.01). Our study suggests genotype-phenotype correlations encouraging further analyses.

High-dose rifampin improves bactericidal activity without increased intracerebral inflammation in animal models of tuberculous meningitis.

Tuberculous meningitis (TB meningitis) is the most severe form of tuberculosis (TB), requiring 12 months of multidrug treatment for cure, and is associated with high morbidity and mortality. High-dose rifampin (35 mg/kg/d) is safe and improves the bactericidal activity of the standard-dose (10 mg/kg/d) rifampin-containing TB regimen in pulmonary TB. However, there are conflicting clinical data regarding its benefit for TB meningitis, where outcomes may also be associated with intracerebral inflammation. We conducted cross-species studies in mice and rabbits, demonstrating that an intensified high-dose rifampin-containing regimen has significantly improved bactericidal activity for TB meningitis over the first-line, standard-dose rifampin regimen, without an increase in intracerebral inflammation. Positron emission tomography in live animals demonstrated spatially compartmentalized, lesion-specific pathology, with postmortem analyses showing discordant brain tissue and cerebrospinal fluid rifampin levels and inflammatory markers. Longitudinal multimodal imaging in the same cohort of animals during TB treatment as well as imaging studies in two cohorts of TB patients demonstrated that spatiotemporal changes in localized blood-brain barrier disruption in TB meningitis are an important driver of rifampin brain exposure. These data provide unique insights into the mechanisms underlying high-dose rifampin in TB meningitis with important implications for developing new antibiotic treatments for infections.

124I-Iodo-DPA-713 Positron Emission Tomography in a Hamster Model of SARS-CoV-2 Infection.

PURPOSE: Molecular imaging has provided unparalleled opportunities to monitor disease processes, although tools for evaluating infection remain limited. Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by lung injury that we sought to model. Activated macrophages/phagocytes have an important role in lung injury, which is responsible for subsequent respiratory failure and death. We performed pulmonary PET/CT with 124I-iodo-DPA-713, a low-molecular-weight pyrazolopyrimidine ligand selectively trapped by activated macrophages cells, to evaluate the local immune response in a hamster model of SARS-CoV-2 infection. PROCEDURES: Pulmonary 124I-iodo-DPA-713 PET/CT was performed in SARS-CoV-2-infected golden Syrian hamsters. CT images were quantified using a custom-built lung segmentation tool. Studies with DPA-713-IRDye680LT and a fluorescent analog of DPA-713 as well as histopathology and flow cytometry were performed on post-mortem tissues. RESULTS: Infected hamsters were imaged at the peak of inflammatory lung disease (7 days post-infection). Quantitative CT analysis was successful for all scans and demonstrated worse pulmonary disease in male versus female animals (P < 0.01). Increased 124I-iodo-DPA-713 PET activity co-localized with the pneumonic lesions. Additionally, higher pulmonary 124I-iodo-DPA-713 PET activity was noted in male versus female hamsters (P = 0.02). DPA-713-IRDye680LT also localized to the pneumonic lesions. Flow cytometry demonstrated a higher percentage of myeloid and CD11b + cells (macrophages, phagocytes) in male versus female lung tissues (P = 0.02). CONCLUSION: 124I-Iodo-DPA-713 accumulates within pneumonic lesions in a hamster model of SARS-CoV-2 infection. As a novel molecular imaging tool, 124I-Iodo-DPA-713 PET could serve as a noninvasive, clinically translatable approach to monitor SARS-CoV-2-associated pulmonary inflammation and expedite the development of novel therapeutics for COVID-19.

Pathogens Detection in the Small Hive Beetle (Aethina tumida (Coleoptera: Nitidulidae)).

Aethina tumida Murray is currently a worldwide emergent pest of Apis mellifera L. hives. Although the damaging effect on the colony stores and brood is well known, the possible role of these beetles as a disease carrier is not clear. This is the first report of DNA presence of the trypanosome honeybee parasite Lotmaria passim and Crithidia bombi, and the Apis mellifera filamentous virus (AmFV) in A. tumida. Further studies will be needed to determine if A. tumida is indeed a mechanical or biological vector of these pathogens.

Correction to: Osteopontin/secreted phosphoprotein-1 behaves as a molecular brake regulating the neuroinflammatory response to chronic viral infection.

An amendment to this paper has been published and can be accessed via the original article.

Osteopontin/secreted phosphoprotein-1 behaves as a molecular brake regulating the neuroinflammatory response to chronic viral infection.

BACKGROUND: Osteopontin (OPN) as a secreted signaling protein is dramatically induced in response to cellular injury and neurodegeneration. Microglial inflammatory responses in the brain are tightly associated with the neuropathologic hallmarks of neurodegenerative disease, but understanding of the molecular mechanisms remains in several contexts poorly understood. METHODS: Micro-positron emission tomography (PET) neuroimaging using radioligands to detect increased expression of the translocator protein (TSPO) receptor in the brain is a non-invasive tool used to track neuroinflammation in living mammals. RESULTS: In humanized, chronically HIV-infected female mice in which OPN expression was knocked down with functional aptamers, uptake of TSPO radioligand DPA-713 was markedly upregulated in the cortex, olfactory bulb, basal forebrain, hypothalamus, and central grey matter compared to controls. Microglia immunoreactive for Iba-1 were more abundant in some HIV-infected mice, but overall, the differences were not significant between groups. TSPO+ microglia were readily detected by immunolabeling of post-mortem brain tissue and unexpectedly, two types of neurons also selectively stained positive for TSPO. The reactive cells were the specialized neurons of the cerebellum, Purkinje cells, and a subset of tyrosine hydroxylase-positive neurons of the substantia nigra. CONCLUSIONS: In female mice with wild-type levels of osteopontin, increased levels of TSPO ligand uptake in the brain was seen in animals with the highest levels of persistent HIV replication. In contrast, in mice with lower levels of osteopontin, the highest levels of TSPO uptake was seen, in mice with relatively low levels of persistent infection. These findings suggest that osteopontin may act as a molecular brake regulating in the brain, the inflammatory response to HIV infection.

Osteopontin and Integrin Mediated Modulation of Post-Synapses in HIV Envelope Glycoprotein Exposed Hippocampal Neurons.

The advent of Human Immunodeficiency Virus (HIV) antiretrovirals have reduced the severity of HIV related neurological comorbidities but they nevertheless remain prevalent. Synaptic degeneration due to the action of several viral factors released from infected brain myeloid and glia cells and inflammatory cytokines has been attributed to the manifestation of a range of cognitive and behavioral deficits. The contributions of specific pro-inflammatory factors and their interplay with viral factors in the setting of treatment and persistence are incompletely understood. Exposure of neurons to chemokine receptor-4(CXCR4)-tropic HIV-1 envelope glycoprotein (Env) can lead to post-synaptic degradation of dendritic spines. The contribution of members of the extracellular matrix (ECM) and specifically, of perineuronal nets (PNN) toward synaptic degeneration, is not fully known, even though these structures are found to be disrupted in post-mortem HIV-infected brains. Osteopontin (Opn, gene name SPP1), a cytokine-like protein, is found in abundance in the HIV-infected brain. In this study, we investigated the role of Opn and its ECM integrin receptors, ß1- and ß3 integrin in modifying neuronal synaptic sculpting. We found that in hippocampal neurons incubated with HIV-1 Env protein and recombinant Opn, post-synaptic-95 (PSD-95) puncta were significantly increased and distributed to dendritic spines when compared to Env-only treated neurons. This effect was mediated through ß3 integrin, as silencing of this receptor abrogated the increase in post-synaptic spines. Silencing of ß1 integrin, however, did not block the increase of post-synaptic spines in hippocampal cultures treated with Opn. However, a decrease in the PNN to ßIII-tubulin ratio was found, indicating an increased capacity to support spine growth. From these results, we conclude that one of the mechanisms by which Opn counters the damaging impact of the HIV Env protein on hippocampal post-synaptic plasticity is through complex interactions between Opn and components of the ECM which activate downstream protective signaling pathways that help maintain the potential for effective post-synaptic plasticity.

Investigation of influence of growing pigs' positive affective state on behavioral and physiological parameters using structural equation modeling.

The aim of the present study was to investigate whether the primarily positive affective state of fattening pigs influences various behavioral and physiological parameters such as the pigs' playing behavior, way of behaving in behavioral tests, body language signals, or diameter, and astroglia cell numbers of hippocampi, salivary immunoglobulin A (IgA) content, or salivary protein composition. Additionally, the suitability of the variables mentioned was examined to assess the pigs' positive affective state in practice, which still constitutes a latent variable not itself measurable. For this, a dataset including behavioral and physiological data of 60 fattening pigs from 3 different farms with different housing systems was analyzed by the partial least squares structural equation modeling (PLS-SEM) method. A hierarchical component model (HCM) was used including the pigs' positive affective state as a higher-order component (HOC) and the behavioral and physiological parameters as lower-order components (LOC). Playing behavior, body language signals, and behavioral tests were revealed, in this order, to be most influenced by the pigs' positive affective state since these resulted in the corresponding path coefficients (PC) of PC = 0.83, PC = 0.79, and PC = 0.62, respectively. Additionally moderate and weak R2-values occurred for the endogenous latent variables playing behavior (R2 = 69.8%), body language signals (R2 = 62.7%), and behavioral tests (R2 = 39.5%). Furthermore, the indicator of the "locomotor play" showed the highest indicator reliability (IR) (IR = 0.85) to estimate the latent variable of pigs' positive affective state. The results of the present study supplement the comprehension and assessment of the pigs' positive affective state in general.

Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris.

AIMS: Isolate and characterize a laccase-encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential. METHODS AND RESULTS: Lac I was cloned and sequenced, it contains 2447 pb obtained by PCR and long-distance inverse PCR. Upstream of the structural region of the laccase gene, response elements such as metals, antioxidants, copper, nitrogen and heat shock were found. The coding region consisted of a 1563-pb ORF encoding 521 amino acids. Lac I was functionally expressed in P. pastoris and it was shown that the gene cloned using the α-factor signal peptide was more efficient than the native signal sequence, in directing the secretion of the recombinant protein. Km and highest kcat /Km values towards ABTS, followed by 2,6-dimethylphenol, were similar to other laccases. Lac I showed tolerance to NaCl and solvents, and nine synthetic dyes could be degraded to different degrees. CONCLUSIONS: Lac I-encoding gene could be successfully sequenced having cis-acting elements located at the regulatory region. It was found that lac I cDNA expressed in P. pastoris using the α-factor signal peptide was more efficient than the native signal sequence. The purified Lac I exhibited high tolerance towards NaCl and various solvents and degraded some recalcitrant synthetic dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The cis-acting elements may be involved in the transcriptional regulation of laccase gene expression. These results may provide a further insight into potential ways of optimizing fermentation process and also open new frontiers for engineering strong promoters for laccase production. The Lac I stability in chloride and solvents and broad decolorization of synthetic dyes are important for its use in organic synthesis work and degradation of dyes from textile effluents respectively.

Cross-cultural adaptation and validation of the Social Comfort Questionnaire for Brazilian adult survivors of burns.

PURPOSE: Burn patients may encounter social barriers and stigmatization. The objectives of this study were to adapt the Social Comfort Questionnaire (SCQ) into Brazilian Portuguese and to assess the psychometric properties of the adapted version. METHODS: Cross-cultural adaptation of the 8 items of the SCQ followed international guidelines. We interviewed 240 burn patients and verified the SCQ internal consistency, test-retest reliability and construct validity, correlating the scores with depression [Beck Depression Inventory (BDI)], affect/body image and interpersonal relationships [Burns Specific Health Scale-Revised (BSHS-R)] and self-esteem [Rosenberg's Self-Esteem Scale (RSES)]. We also performed a confirmatory factor analysis (CFA). RESULTS: The cross-cultural adaptation resulted in minor semantic modifications to the original SCQ version. After CFA, a reduced 6-item version showed satisfactory fit to the one-factor model (RMSEA = 0.05, CFI = 0.99, TLI = 0.99). Cronbach alpha's was 0.80, and test-retest intraclass correlation coefficient was 0.86. The final version presented a strong negative correlation with depression (BDI), and strong positive correlations with affect/body image (BSHS-R), interpersonal relationships (BSHS-R) and self-esteem (RSES) (all p < 0.001). CONCLUSION: The results showed that the SCQ Brazilian Portuguese adapted version complies with the validity and reliability criteria required for an instrument assessing social comfort in Brazilian burn patients. The Brazilian version yields a single score that is easy to interpret and well understood by patients.

Assessment of bevacizumab quality and stability in repackaged syringes for clinical use.

OBJECTIVES: Among measures taken to optimise financial resources, the off-label use of bevacizumab (Avastin) in the treatment of age-related macular degeneration (AMD) involves its repackaging from higher volume dosage forms. This use requires studies to analyse the viability of the repackaged preparations to ensure their quality, safety and efficacy. Our aim was to assess the structural stability and particle size of bevacizumab after it was repackaged from the original glass vials and stored in plastic syringes. METHODS: High performance liquid chromatography by size exclusion (HPLC-SE) was used to quantify the bevacizumab and determine its degradation products after stress stability testing, with a particle size counter employed after repackaging and subsequent storage. RESULTS: The syringes stored for 3 days at 4°C maintained the area of the main chromatographic peak above 100±10% of its initial value, and the observed particle size is the same as at baseline (20 nm) but with a double distribution towards larger sizes. CONCLUSIONS: This study shows how the repackaging of Avastin in plastic syringes permits their use for 3 days if stored under normal refrigeration. In this way, hospital pharmacy services can help optimise health resources without compromising the pharmaceutical standards of the drug.

Study of quality and stability of ursodeoxycholic acid formulations for oral pediatric administration.

This paper describes a rational method of characterizing the biopharmaceutical stability of two oral suspensions of ursodeoxycholic acid (UDCA) used in pediatrics. Because there is no commercial presentation of UDCA that can administer appropriate doses for infants and children, an active pharmaceutical ingredient (API) formulation is required. Due to its very low solubility and low dose in the formula (1.5%), two different suspensions with minimal use of excipients were studied, avoiding the use of complex additives and those not recommended by the European Medicines Agency (EMA). Adherence to Standard Operating Procedure (SOP) allows the preparation of formulations with appropriately sized and stable particles, and suitable rheological behavior in withdrawing the dose after stirring. Dose uniformity, expressed as mass and content variability, was determined using the criteria of the European and the United States Pharmacopoeia. Additionally, dose content variation of every mass determined was studied. A rational method was developed for determining the dose uniformity of UDCA in suspensions, whether freshly prepared or after storage under different conditions for 30 and 60 days. This method permits detection of differences between doses taken at different heights in the vessel at various times and storage conditions. UDCA was stable under all conditions studied, requiring the presence of glycerol in the formulation to obtain the declared API value after stirring. Storage of UDCA suspensions in a refrigerator increased variability between doses.

The influence of environmental factors on the abundance and recruitment of the sand crab Emerita analoga (Stimpson 1857): source-sink dynamics?

The sandcrab Emerita analoga is the dominant species inhabiting sandy beaches along the Pacific coast of the American continent. In our study, 10 sandy beaches were sampled seasonally from 2006 to 2011, including coastal planktonic sampling from 2006 to 2008. Two major population cores were detected, the first one in the northern part of the study area and the second in the area immediately to the south of the Itata River mouth. Zoeal stages were found along the entire coastal zone. Highest densities and recruitment were found during spring and summer of each year. PLS regression indicated that source-sink habitat proxies correlated positively with morphodynamic parameters; while beach slope and total organic matter were negatively correlated. These results agree with the source-sink hypothesis, finding higher densities of adults, recruits and cohort recurrence on open coast beaches with milder physical dynamics. Furthermore, a hypoxic event and a mega-earthquake/tsunami negatively affected recruitment at the inter-annual scale.

Design of a pediatric oral formulation with a low proportion of hydrochlorothiazide.

It is a normal pediatric practice in community and hospital pharmacies to prepare a new drug formulation when no commercial forms of it are available. Any dose or stability control is usually done for these types of compounding formulations due to the effort which means to develop these types of tests in pharmacies. We have studied five different hydrochlorothiazide oral formulations prepared with traditional compounding techniques in pharmacies to treat heart failure and edemas in babies. A Standard Operating Procedure (SOP) was done for every suspension. After the strictly monitoring of the SOP, every suspension was subjected to quality control tests (pH, particle size, viscosity, dose content and stability). There is only one studied formulation that guarantees the correct dose administering and stability after 3 weeks stored at 5 °C and light protected. Both, the percentage of wetting agent and the viscosity of the suspensor vehicle in this formulation make the correct dose administering possible after the formulation is shaken.

Quality control and physical and chemical stability of hydrocortisone oral suspension: an interlaboratory study.

There were two objectives of this study, one of which was to ensure that different 1-mg/mL hydrocortisone oral suspensions that were prepared as compounded preparations in four accredited laboratories using the same standard operating procedure were in accordance with the general quality controls of these disperse systems and the specific requirements of the European Pharmacopeia, the Royal Spanish Pharmacopeia, and the United States Pharmacopeia. The other objective was to confirm the shelf-life of 90 days that was established for this type of compounded preparation. All the suspensions were in accordance with the organoleptic controls and with the theoretically calculated volume per unit of the preparation, but the amount of active ingredient per volume of the preparations were smaller than the theoretically expected. This result was attributed to the standard operating procedure because no significative differences between laboratories and days were detected, in which an excess of active ingredient is necessary to include in the process to ensure the declared dose. By high-performance liquid chromatography, it was possible to confirm the 90 days of stability of the drug at 5 degrees Celsius.

High-voltage electrical burn injuries: functional upper extremity assessment.

High-voltage electric injuries have many manifestations, and an important complication is the damage of the central/peripheral nervous system. The purpose of this work was to assess the upper limb dysfunction in patients injured by high-voltage current. The evaluation consisted of analysis of patients' records, cutaneous-sensibility threshold, handgrip and pinch strength and a specific questionnaire about upper limb dysfunctions (DASH) in 18 subjects. All subjects were men; the average age at the time of the injury was 38 years. Of these, 72% changed job/retired after the injury. The current entrance was the hand in 94% and grounding in the lower limb in 78%. The average burned surface area (BSA) was 8.6%. The handgrip strength of the injured limb was reduced (p<0.05) and so also that of the three pinch types. The relationship between the handgrip strength and the DASH was statistically significant (p<0.001) as well as the relationship between the three pinch types (p