Bacteriophage genome engineering with CRISPR-Cas13a.

Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.

A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.

All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently1. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.

Cas13 Helps Bacteria Play Dead when the Enemy Strikes.

How RNA-targeting CRISPR-Cas13 functions as a phage defense system has been mysterious. Recently in Nature, Meeske et al. (2019) demonstrate that Cas13 provides potent immunity to dsDNA phages without cutting their genome. By sensing phage transcripts and destroying RNA nonspecifically to arrest the cell into dormancy, Cas13 provides herd immunity.

Effects of bilateral breast reduction on peak airway pressure and pulmonary function tests.

PURPOSE: Surgical reduction due to breast size is not carried out merely for anesthetic concerns but also for such complaints as breast pain belonging to breast and skeletal system, back pain, neck pain, and intertriginous rashes. This study aims to investigate the effect of bilateral breast reduction surgery on maximum inspiratory pressure (Ppeak) and pulmonary functions. This study aims to investigate the effect of bilateral breast reduction surgery on pulmonary function test. METHODS: The study included 50 patients who would undergo bilateral breast reduction. Patients were divided into two groups: group II were given positive end-expiratory pressure (PEEP), which was not administered to the group I. Patients were checked in terms of maximum inspiratory pressures (Ppeak) before surgery, after first and second breasts were removed, and after surgery. Pulmonary function tests were carried out on preoperative, postoperative second and 14th days. As RFT, forced vital capacity (FVC), FEV1 (forced expiratory volume at the first second of FVC), FEV1/FVC and PEF (peak expiratory flow rate) were measured. RESULTS: In both groups, demographic data were not found to statistically significant differences (P > 0.05). When compared both groups in terms of preoperative FVC and FEV1/FVC 14th day, a significant increase was found on the 14th day (P < 0.05). A significant difference was not established between groups in terms of Ppeak values (P > 0.05). Ppeak was found to be significantly higher in group I (22.28 ± 7.56) at the end of intubation compared with group II (19.04 ± 3.73) (P = 0.002, P < 0.05). Similarly, preoperative Ppeak was established to be 21.88 ± 7.51 in group I and it was significantly higher compared with group II (19.44 ± 4.08), (P = 0.002, P < 0.05). When compared Ppeak values at the end of intubation and before operation with entry values a statistically significant difference was not found in either group (P = 0.76, P > 0.05). CONCLUSIONS: Some researchers reported a positive correlation between FVC, FEV1/FVC, and PEF along with the excised tissue mass. We established a positive correlation between the excised tissue weight and FVC and FEV1/FVC and that PEEP application did not have an impact on Ppeak.

How bacteria control the CRISPR-Cas arsenal.

CRISPR-Cas systems are adaptive immune systems that protect their hosts from predation by bacteriophages (phages) and parasitism by other mobile genetic elements (MGEs). Given the potent nuclease activity of CRISPR effectors, these enzymes must be carefully regulated to minimize toxicity and maximize anti-phage immunity. While attention has been given to the transcriptional regulation of these systems (reviewed in [1]), less consideration has been given to the crucial post-translational processes that govern enzyme activation and inactivation. Here, we review recent findings that describe how Cas nucleases are controlled in diverse systems to provide a robust anti-viral response while limiting auto-immunity. We also draw comparisons to a distinct bacterial immune system, restriction-modification.

Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency.

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that prp8 alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of prp8 alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a prp2 mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that prp8 RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Effects of polyphenols on skin damage due to ultraviolet A rays: an experimental study on rats.

BACKGROUND: Ultraviolet (UV) radiation causes many acute and chronic conditions such as oedema of the skin, sunburn, immunosuppression, photo-ageing and skin cancer. The use of antioxidants has become of paramount importance in prevention of the damage caused by ultraviolet radiation. Epigallocatechin-3-gallate (EGCG), one of the main components of green tea, has been reported to have anti-inflammatory, antioxidant and anticarcinogenic properties. AIM: The aim of this experimental study was to investigate to what extent EGCG prevented acute skin damage caused by UVA. MATERIAL AND METHOD: The sample contained 2% EGCG, which was prepared in hydrophilic ointment (USP XXIV) as the vehicle. Twenty-four 12-week-old Wistar albino rats are included in the study and divided into four groups, each containing six rats. Group I was formed to be the control group, which was not applied any topical medication or exposed to UV radiation. Group II was formed to observe acute effects of UVA on the skin, Group III was formed to observe effectiveness of topical EGCG on the skin applied 30 min after exposure to UVA, and Group IV was formed to observe topical EGCG applied 30 min before exposure to UVA. All groups were examined for sunburn cells, leucocyte infiltration, dermo-epidermal activity, collagen changes and elastic fibre pathologies on 24 and 72 h. Statistical analysis was performed using spss 11.5, and chi-squared test was used for the evaluation of parameters. RESULTS: Group IV showed a statistically significant decrease in sunburn cells and dermo-epidermal activation compared with Group II. Group II showed significant increase in all parameters compared with Group I, showing the effects of UV exposure alone, and no difference was detected in Group II and III. CONCLUSION: These results show a protective effect of EGCG when applied topically before UVA exposure. No benefit was detected when EGCG was applied after UV exposure.

Linfoma Não-Hodgkin experiência de um Centro de Referência / Non-Hodkin Lynphoma experience of a Reference Center

Verificar a ocorrência de casos novos de Linfoma Não-Hodgkin LNH nas crianças e adolescentes diagnosticados em um Centro de Referência de Santa Catarina, em relação a sexo, cor ou raça, idade, manifestações clínicas, procedência, extensão clínica da doença e status vital. Método: estudo observacional, descritivo e longitudinal realizado no Serviço de Onco-Hematologia do Hospital Infantil Joana de Gusmão. A população de estudo constituiu-se de todos os casos novos de crianças e adolescentes com diagnóstico histopatológico de LNH, atendidos durante o período de janeiro de 1997 a dezembro de 2001 neste hospital. Resultados: foram registrados vinte e quatro casos novos de LNH em crianças e adolescentes no período de estudo. O LNH ocorreu no sexo masculino em 75% dos casos, no pré-escolar em 50% e na mesorregião de procedência Sul Catarinense em 29,1%. Observou-se ao diagnóstico doença não-localizada em 91,7% dos casos (estádios III e IV). A localização primária do tumor foi o abdome em 62,4%. As manifestações gastro-intestinais estavam presentes em 70% dos casos. O tipo histológico de pequenas células não-clivadas ocorreu em 54,2% dos casos. Ao final do estudo, 75% dos pacientes estavam vivos. Conclusão: o LNH é mais comum no pré-escolar, no sexo masculino e na mesorregião de procedência Sul Catarinense. A localização primária do tumor mais freqüente é o abdome. Há predomínio das manifestações clínicas gastro-intestinais e da doença não localizada no diagnóstico. O linfoma de pequenas células não-clivadas é o tipo histológico predominante. A maioria das crianças e adolescentes portadores de LNH está viva e fora de tratamento...

Multiple atrophies following tooth extraction.

Following a molar tooth extraction, the patient developed a depression and atrophy of the right ear, the right half of the mentum, and the tongue. These atrophy deformities were successfully corrected with dermografts.

Use of beta blockers for increasing survival of free fat grafts.

Fat tissue is abundant in the body and preferred in soft tissue augmentation. Since resorption is its greatest disadvantage, many agents have been tried in an attempt to increase fat graft survival in various studies. Yet selective beta1 blocker had not been used before. The effect of selective beta1 blockers on resorption following auto-transplantation of free fat graft was examined in rats. The effect of selective beta1 blockers was compared with those of insulin and saline solution as a control. For this comparison, the weight of fat grafts was measured both during and nine months after operation using the "liquid overflow method" in which obtained material was put into 5 cc syringe filled with saline solution and the increase in liquid level was recorded as graft volume. Histological examination of grafts and statistical analyses of the results were also made. Our in vivo study demonstrated that selective beta1 blocker significantly increased free fat graft survival. It is our conclusion that selective beta1 blocker is a promising agent for decreasing fat graft resorption.

[A model of extreme insulin resistance with carbohydrate intolerance in a patient with Werner's syndrome]. / Modelo de insulinorresistencia extrema con intolerancia hidrocarbonada en un paciente con el síndrome de Werner.

We present the study of a patient suffering insulin resistance with Werner's syndrome which had abnormal glucose tolerance determined by euglycemic fixing ("Clamp") using an artificial pancreas (Biostator GCIIS, Miles Martin) and the binding of insulin to its receptor in erythrocytes. The results obtained show a dose-response curve of insulin serum levels to dextrose infusion rate, shifted significantly to the right and bottom which indicates a diminished insulin sensitivity; similarly a moderate decrease in receptor is obtained with no decrease in their affinity and absence of abnormalities in contraregulatory hormones. These results are compatible with a postreceptor alteration.

Severe hypoglycemia as a short-term side-effect of the somatostatin analog SMS 201-995 in insulin-dependent diabetes mellitus.

We report on a case of severe hypoglycemia observed after injection of the somatostatin analog SMS 201-995 in an insulin-dependent diabetic patient connected to an artificial pancreas (Biostator GCIIS). Hypoglycemia occurred in a sudden and unexpected way 23 min after the first SMS injection (50 ug). Details on patient's characteristics and on the peculiar clinical situation in which this complication appeared are given. Furthermore, the possible mechanism(s) responsible for the hypoglycemia are thoroughly discussed. Finally we express a call for caution when administering this drug in insulin-dependent diabetes mellitus.

Efectos extrapancreáticos de las sulfonilureas / Extrapancreatic effects of sulfonylureas

Las sulfonilureas, en particular las de segunda generación, son objeto de un creciente interés debido principalmente a que hoy en día se está comenzando a comprender la fisiopatología de la Diabetes Mellitus no Insulinodependiente (DMNID). La acción hipoglucemiante producida por estos fármacos a corto plazo es conocida desde hace tiempoñ sin embargo su acción a largo plazo no está totalmente clara. Varios son los mecanismos invocados, pero probablemente sólo dos de ellos están involucrados: la potenciación de la acción de la insulina a nivel periférico y la disminución de la producción hepática de glucosa. La potenciación de la acción de la insulina se realiza a nivel posreceptor, teniendo como principal consecuencia una disminución de la insulinorresistencia presente en los pacientes con DMNID. El mecanismo por el cual se produce la disminución de la producción hepatica de glucosa no está tan bien clarificado como el anterior. Sin embargo se ha observado una variación en unn metabolito intracelular, la fructosa 2,6-difosfato, que podría ser uno de los mecanismos subyacentes en los efectos posreceptor de aquellas sulfonilureas

Efectos extrapancreáticos de las sulfonilureas / Extrapancreatic effects of sulfonylureas

Las sulfonilureas, en particular las de segunda generación, son objeto de un creciente interés debido principalmente a que hoy en día se está comenzando a comprender la fisiopatología de la Diabetes Mellitus no Insulinodependiente (DMNID). La acción hipoglucemiante producida por estos fármacos a corto plazo es conocida desde hace tiempoñ sin embargo su acción a largo plazo no está totalmente clara. Varios son los mecanismos invocados, pero probablemente sólo dos de ellos están involucrados: la potenciación de la acción de la insulina a nivel periférico y la disminución de la producción hepática de glucosa. La potenciación de la acción de la insulina se realiza a nivel posreceptor, teniendo como principal consecuencia una disminución de la insulinorresistencia presente en los pacientes con DMNID. El mecanismo por el cual se produce la disminución de la producción hepatica de glucosa no está tan bien clarificado como el anterior. Sin embargo se ha observado una variación en unn metabolito intracelular, la fructosa 2,6-difosfato, que podría ser uno de los mecanismos subyacentes en los efectos posreceptor de aquellas sulfonilureas (AU)

Effect of a long-acting somatostatin derivative SMS 201-995 (sandostatin) on glucose homeostasis in type I diabetes mellitus.

The infusion of natural somatostatin (SRIF) has been able to partially correct postprandial hyperglycemic reactions in insulin-dependent diabetes mellitus (IDDM). SMS 201-995 (Sandostatin) is a long-acting derivative with a growth hormone-suppressive effect 10-60 times more potent than the native peptide. The effect of SMS 201-995 (50 micrograms s.c.) on glucose control by exogenous insulin has been documented in a series of type I diabetics after stabilization of blood sugar by an artificial pancreas. Inhibition of counterregulatory mechanisms significantly diminished the postprandial hyperglycemia, and insulin requirements, both total and 2 h after meals, were markedly decreased. Also the effect of a single s.c. injection of 100 micrograms SMS 201-995 on the dawn phenomenon in a patient with poorly adjustable diabetes was investigated. The glucose escape observed during the control night was blocked by SMS 201-995. Thus, the stabilizing action of this peptide on postprandial and nocturnal hyperglycemia in unstable diabetes warrants further studies.