The exo-ß-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases.

Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

Impact of Physician Inspection in the Detection of Phlebitis and Factors Contributing to it in Admitted Children of a Tertiary Care Hospital: A Prospective Study.

OBJECTIVE: To determine whether additional dedicated observations by a doctor would increase the pickup rate of phlebitis and related complications due to intravenous cannulation. Also, to identify the common demographic and clinical factors predisposing to phlebitis in children. METHODS: This prospective study was conducted from January 2019 through December 2019. A total of 184 children with 341 cannulations were enrolled. The authors added dedicated inspection of cannula by the physician during rounds in attempt to increase the detection of phlebitis. The difference in detection rates of thrombophlebitis by physician and routine observation along with the demographic and clinical variables contributing to thrombophlebitis in hospitalized children were studied. RESULTS: Total incidence of thrombophlebitis was 35/341 (10.3%), which included 2 extravasations. Nineteen events (55%) were picked up by routine nursing observations and an additional 16 (45%) by the physician. Among the factors contributing to phlebitis, maternal education status <12th standard (41.7% vs. 15.1% P = 0.018), cannula located on forearm (p value 0.008), bigger cannula size (18% vs. 8.6% P = 0.008), non-splinted cannula (14.4% vs. 7.7% P = 0.046) intravenous fluid containing potassium (15.4% vs. 2.9%, P = 0.001) and concentrated infusions in particular aminophylline and magnesium sulphate (26.3% vs. 9.3%, P = 0.018) were found to be significantly associated with the development of phlebitis. CONCLUSIONS: Physician inspection during rounds can improve phlebitis detection and will also increase the sensitivity of nursing observation.

Bacteria's different ways to recycle their own cell wall.

The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This "anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.

Recovery of the Peptidoglycan Turnover Product Released by the Autolysin Atl in Staphylococcus aureus Involves the Phosphotransferase System Transporter MurP and the Novel 6-phospho-N-acetylmuramidase MupG.

The peptidoglycan of the bacterial cell wall undergoes a permanent turnover during cell growth and differentiation. In the Gram-positive pathogen Staphylococcus aureus, the major peptidoglycan hydrolase Atl is required for accurate cell division, daughter cell separation and autolysis. Atl is a bifunctional N-acetylmuramoyl-L-alanine amidase/endo-ß-N-acetylglucosaminidase that releases peptides and the disaccharide N-acetylmuramic acid-ß-1,4-N-acetylglucosamine (MurNAc-GlcNAc) from the peptido-glycan. Here we revealed the recycling pathway of the cell wall turnover product MurNAc-GlcNAc in S. aureus. The latter disaccharide is internalized and concomitantly phosphorylated by the phosphotransferase system (PTS) transporter MurP, which had been implicated previously in the uptake and phosphorylation of MurNAc. Since MurP mutant cells accumulate MurNAc-GlcNAc and not MurNAc in the culture medium during growth, the disaccharide represents the physiological substrate of the PTS transporter. We further identified and characterized a novel 6-phospho-N-acetylmuramidase, named MupG, which intracellularly hydrolyses MurNAc 6-phosphate-GlcNAc, the product of MurP-uptake and phosphorylation, yielding MurNAc 6-phosphate and GlcNAc. MupG is the first characterized representative of a novel family of glycosidases containing domain of unknown function 871 (DUF871). The corresponding gene mupG (SAUSA300_0192) of S. aureus strain USA300 is the first gene within a putative operon that also includes genes encoding the MurNAc 6-phosphate etherase MurQ, MurP, and the putative transcriptional regulator MurR. Using mass spectrometry, we observed cytoplasmic accumulation of MurNAc 6-phosphate-GlcNAc in ΔmupG and ΔmupGmurQ markerless non-polar deletion mutants, but not in the wild type or in the complemented ΔmupG strain. MurNAc 6-phosphate-GlcNAc levels in the mutants increased during stationary phase, in accordance with previous observations regarding peptidoglycan recycling in S. aureus.

Términos económicos más utilizados en la salud (II parte) / Most used economic terms in health (II part)

En este artículo se ofrece el resto de los términos económicos, organizados alfabéticamente de la letra E a la Z, con lo cual se completa el glosario iniciado en el número anterior de esta revista. Se espera que pueda ser útil a todos los estudiantes y profesionales de las ciencias médicas que lo necesiten como material de consulta, y para todos aquellos lectores que deseen incrementar su acervo cultural en la esfera económica.

In this work the rest of the economic terms alphabetically organized are offered from E to Z letters, with which the glossary initiated in the previous number of the present journal is completed. It is expected that this glossary can be useful to all the students and professionals of the medical sciences that need it as consultation material, and for all those readers that want to increase their cultural heritage in the economic sphere.

Términos económicos más utilizados en la salud (II parte) / Most used economic terms in health

Se ofrecen 525 términos económicos organizados alfabéticamente, cuya mayoría comúnmente se emplean en la salud; algunos han sido actualizados por los autores según los cambios de la economía en los momentos actuales, lo que puede contribuir a enriquecer la cultura de estudiantes, profesores y trabajadores no vinculados directamente a la esfera económica, pero que indirectamente participan de ella. El empleo de una serie de vocablos en los Lineamientos de la Política Económica y Social del Partido y la Revolución aprobados en el VI Congreso del Partido Comunista de Cuba, que no eran de uso común en este país por su diferencia socioeconómica con la economía imperante en el mundo, constituyó el motivo fundamental para la confección de este glosario.

Five hundred and twenty five economic terms alphabetically organized are offered, the majority of them are commonly used in health; some of them have been updated by the authors according to the changes of economy in the current moments, which can contribute to enrich the culture of students, professors and workers not directly linked to the economic sphere, but that participate of it indirectly. The use of a series of words in the Guidelines of the Party and Revolution Economic and Social Politics approved in the VI Congress of the Cuban Communist Party, that were not commonly used in this country due to its socioeconomic difference with the prevailing economy in the world, constituted the fundamental reason for making this glossary.

Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (ßNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to ßNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

DESIGN OF A SIMPLE SLOW COOLING DEVICE FOR CRYOPRESERVATION OF SMALL BIOLOGICAL SAMPLES.

BACKGROUND: Slow cooling is a cryopreservation methodology where samples are cooled to its storage temperature at controlled cooling rates. OBJECTIVE: Design, construction and evaluation of a simple and low cost device for slow cooling of small biological samples. MATERIALS AND METHODS: The device was constructed based on Pye's freezer idea. A Dewar flask filled with liquid nitrogen was used as heat sink and a methanol bath containing the sample was cooled at constant rates using copper bars as heat conductor. RESULTS: Sample temperature may be lowered at controlled cooling rate (ranging from 0.4°C/min to 6.0°C/min) down to ~-60°C, where it could be conserved at lower temperatures. An example involving the cryopreservation of Neuro-2A cell line showed a marked influence of cooling rate over post preservation cell viability with optimal values between 2.6 and 4.6°C/min. CONCLUSION: The cooling device proved to be a valuable alternative to more expensive systems allowing the assessment of different cooling rates to evaluate the optimal condition for cryopreservation of such samples.

La Economía y su relación con la salud de la población cubana / Economy and its relation to the health of the Cuban population

Se realizó una investigación observacional y descriptiva para mostrar las derivaciones de la relación economía/salud mediante datos demográficos, epidemiológicos y socioeconómicos, vinculados al proceso salud-enfermedad, sobre todo en la provincia de Santiago de Cuba durante el bienio 2010-2011. Entre los principales resultados sobresalieron, además del incremento de la población cubana en 2011, de la tasa anual de crecimiento, del grado de envejecimiento y de los gastos en salud y asistencia, que 62,4 % de la población económicamente activa de Cuba estaba compuesta por hombres y 37,6% por mujeres, con mayor porcentaje de desocupación de estas últimas en comparación con los primeros (3,5 y 3,0, respectivamente). De los valores obtenidos se concluyó que la provincia mostró diferencias en el estado de salud respecto a otras regiones del país, vinculadas con el grado de desarrollo social y el ambiente circundante, así como también que el aumento de los indicadores ya señalados y de las enfermedades crónicas no trasmisibles constituyeron en esa etapa un desafío para el financiamiento de la atención y los programas sanitarios.

An observational and descriptive investigation was carried out to show the derivations of the relation economy/health by means of demographic, epidemiological, social and economic data, linked to the process health-disease, mainly in Santiago de Cuba province during the biennium 2010-2011. Among the main results there were, besides the Cuban population increment in 2011, the annual growth rate, the aging level and the expenses in health and care, that from the economically active population in Cuba, 62.4% was composed by men and 37.6% by women, with higher percentage of unemployment of the last ones in comparison with the first ones (3.5 and 3.0, respectively). From the obtained values it is concluded that the province showed differences in the health condition regarding other domestic regions, linked to the level of social development and the surrounding atmosphere, as well as that the increase of the indicators already mentioned and of the non-communicable chronic diseases constituted in that stage a challenge for the financing of care and sanitary programs.

Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

Bilirubin accumulation and Cyp mRNA expression in selected brain regions of jaundiced Gunn rat pups.

INTRODUCTION: Few data exist on regional brain bilirubin content in the neonatal period when acute bilirubin-induced neurologic damage (BIND) may occur, and no information is available on regional brain expression of cytochrome P450 monooxygenases (Cyps) that oxidize bilirubin. METHODS: Bilirubin content was analyzed by high-performance liquid chromatography and Cyp1a1, 1a2, and 2a3 mRNA expression was analyzed by quantitative PCR (qPCR) in cortex (Cx), cerebellum (Cll), superior colliculi (SC), and inferior colliculi (IC) of 17-d-old hyperbilirubinemic (jj) Gunn rat pups before and after administration of sulphadimethoxine to acutely displace bilirubin from plasma albumin. RESULTS: There was no difference in bilirubin content among brain regions in untreated rats. After intraperitoneal sulphadimethoxine, bilirubin content peaked at fourfold in Cx and SC at 1 h; but at 11- to 13-fold in Cll and IC at 24 h; returning to control levels at 72 h. The Cyp mRNA peaked at 30-70 times control at 1 h in Cx and SC, but at 3-9 times control at 24 h in Cll and IC. DISCUSSION: The close relationship in distinct brain regions between the extent of bilirubin accumulation and induction of mRNA of Cyps suggests Cyps may have a role in protecting selected brain areas from bilirubin neurotoxicity.