Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

Photosensitization in cattle grazing on pastures of Brahciaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis

Aspects of photosensitization in bovines grazing on pastures of Brachiaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures 45(2):117-136, 1978. This paper reports experimental studies on photosensitization in bovines grazing on different pastures of Brachiaria decumbens Stapf in the "Cerrados" region (Planaltina, DF). Climatic conditions, zinc content and occurence of fungi on pastures were investigated. Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures examined. Photosensitization was observed in one animal maintained on a pasture of B. decumbens formed with seeds from Australia. Clinical and necropsy data were similar to those related in literature for sporidesmin-intoxicated animals. An isolate of P. chartarum and samples of bovine bile were assayed for sporidesmin presence.     

Liver transplantation in a patient with cholangiocarcinoma and ulcerative colitis.

A 39 year-old patient with cholangiocarcinoma and pre-existing ulcerative colitis was successfully treated by orthotopic liver transplantation. He was given low doses of prednisone and azathioprine and survived for more than 9 months, dying with tumour metastases, thrombosis of the inferior vena cava and an intra-abdominal abscess. At autopsy the homograft showed little evidence of rejection. Preoperatively the patient had septicemia. Removal of his liver was difficult. The discrepancy between donor and recipient in size of blood vessels and the presence of two hepatic arteries in the donor caused problems during the vascular anastomoses. During the operation cardiac arrest occurred. Postoperatively there were several medical and surgical problems, including intraperitoneal and gastrointestinal hemorrhage, paralysis of the right dome of the diaphragm, sinus bradycardia, massive diuresis, peroneal nerve palsy, and one major and three minor episodes of rejection, which were reversed by giving pulse doses of methylprednisolone intravenously.     

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations.Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.     

MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process—the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor—via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.     

Cryptogenic hepatitis patients have a higher Bartonella sp.-DNA detection in blood and skin samples than patients with non-viral hepatitis of known cause

BackgroundThis study aimed to assess the prevalence of Bartonella sp.-DNA detection in blood and skin samples from patients with non-viral end-stage liver disease awaiting liver transplantation.Methodology/Principal findingsBlood samples and healthy skin fragments from 50 patients were tested using microbiological and molecular methods. Fifteen patients had cryptogenic hepatitis (CH) and 35 had alcoholic, drug-induced or autoimmune liver disease. DNA was extracted from whole blood and liquid culture samples, isolates, and skin fragments. Thirteen of the 50 patients (26%) had Bartonella henselae DNA detection in their blood (9/50) and/or skin (5/50) samples. Colonies were isolated in 3/50 (6%) and infection was detected in 7/50 (14%) of the 50 patients. B. henselae-DNA detection was more prevalent in patients with CH than in other patients (p = 0.040). Of 39 patients followed-up for at least two years, a higher mortality rate was observed among patients with CH infected with B. henselae (p = 0.039).Conclusions/SignificanceFurther studies assessing the role of B. henselae infection in the pathogenesis of hepatitis patients must be urgently conducted.