Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3^CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3^CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3^CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3^CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Cellulose, a linear homopolymer of d-glucopyranose linked by β-1,4-glycosidic bonds, is the major structural component of the cell walls of plants, oomycetes, and algae and constitute the most abundant biopolymer on Earth (1). Cellulose is synthesized by cellulose synthases (CESAs) that belongs to the glycosyltransferase GT-2 superfamily (1, 2). In land plants, cellulose is produced at the plasma membrane by six-lobed rosette-shaped CESA complexes (CSCs) where each CESA is thought to synthesize one cellulose chain (3). The precise number of CESAs per CSC is unresolved but estimated to range between 18 and 36 (4–6).Plants contain multiple cesa genes, with 10 found in the Arabidopsis genome (7). Of these, CESA1, CESA3, CESA6, and the CESA6-like CESAs (i.e., CESA2, CESA5, and CESA9) are involved in primary cell wall formation, whereas CESA4, CESA7, and CESA8 participate in secondary cell wall formation (8–12). These two types of CSCs form heterotrimeric complexes with a ratio of 1:1:1 (13, 14). The Arabidopsis CESAs share an overall sequence identity of ∼60% and have seven transmembrane helices (15). In plants, the catalytic domain (CatD) of the CESAs is located between the second and third transmembrane helices and contains a canonical D, D, D, QxxRW motif (1). While there are similarities between the plant CatD and its counterpart in bacterial cellulose synthases, the CatD is flanked by two plant-specific domains, the so-called plant-conserved region (P-CR) and class-specific region (C-SR) (16). These domains are proposed to have important functions in cellulose synthesis and CESA oligomerization (17).The oligomerization of plant CESAs is thought to be important for the final CSC assembly, and multiple oligomeric states of CESAs, including homodimers, have been reported (18, 19). For example, immunoprecipitation assays using CESA7 fused to a dual His/STRP-tag demonstrated that CESA4, CESA7, and CESA8 could form independent homodimers, and it was hypothesized that the CESA homodimerization may contribute to early stages of CSC assembly. These homodimers might then be converted into CSC heterotrimeric configurations (19). This feature poses a marked difference from the bacterial cellulose synthase complex. However, how CESA homodimers are formed and how they function in cellulose synthesis are unknown.To comprehend the mechanisms behind plant cellulose synthesis, it is essential to acquire structural information about plant CESAs. Indeed, the BcsA–BcsB complex structure from Rhodobacter greatly aided our understanding of the cellulose synthesis in bacteria (20). Nevertheless, there are many differences between bacterial and plant CESAs and the corresponding protein complexes. Extensive efforts have been undertaken to acquire plant CESA structural information, including homology modeling and small-angle X-ray scattering analyses (5, 6, 16, 21, 22). While these efforts have been important to form new hypotheses, they did not reveal significant insights into substrate coordination, cellulose chain extrusion, and complex assembly. Recently, a homotrimeric CESA8 structure from Populus tremula × tremuloides was resolved by cryogenic electron microscopy (cryo-EM), which offered significant new molecular understanding of cellulose microfibril biosynthesis and CESA coordination within the CSC (15). Here we report the crystal structures of Arabidopsis CESA3 CatD (AtCESA3^CatD) in apo and uridine diphosphate (UDP)-glucose (UDP-Glc) bound forms and outline how the CatD might contribute to CESA homodimerization and substrate coordination.     

IL-10 and IL-12p40 in Egyptian patients with HCV-related chronic liver disease

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide with a prevalence of approximately 14% in Egypt. IL-10 is a cytokine produced by Th2 cells. It down-regulates the proinflammatory response and modulates hepatic fibrogenesis. IL-12 is produced by antigen presenting cells. It promotes Th1 cell response and has many antiviral properties. Data concerning the Th-1/Th-2 balance in chronic hepatitis C (CH-C) are rather conflicting. Using ELISA, we assessed serum IL-10 and IL-12p40 levels in 66 Egyptian patients with HCV-related liver illness (CH-C, cirrhosis, and HCC), and their relationship to disease activity. Our results showed that spontaneous IL-10 was undetectable in patients with CH-C, HCC or controls. Only 5/22 (23%) of patients with cirrhosis showed detectable levels of IL-10. IL-12p40 was elevated in the patient groups compared to controls (p= 0.01, p= 0.01, p= 0.05 in CH-C, cirrhosis and HCC, respectively). The presence of IL-12p40 was associated with HCV level of viremia and serum AST. Serum ALT level was significantly associated with the level of IL-12p40. IL-12p40 was unrelated to liver histology or fibrosis. We concluded that in the Egyptian patients an augmentation of IL-12p40 and a suppression of IL-10 are both found. Whether this pattern is related to HCV genotype 4, or to the presence of schistosomiasis would need to be further investigated.     

Ultrasonic energy. Effects on vascular function and integrity

BACKGROUND. Ultrasonic energy transmitted via flexible wire probes provides a new means of ablating atherosclerotic plaque. We studied the effects of ultrasonic energy (20 kHz) delivered via a ball-tipped wire probe on arterial vasomotor behavior in rabbit thoracic aortas in a perfused whole-vessel model. METHODS AND RESULTS. After precontraction with phenylephrine (10(-5) M) or KCl (60 mM), the effects of ultrasonic energy (0.7-5.5 W x 60 seconds, 42-330 J) on arterial vasomotor behavior were measured using long-axis ultrasonic vessel imaging of the proximal (ultrasonic probe-treated) and distal (untreated) control segments. The efficacy of plaque ablation at these same probe-tip power outputs was evaluated in atherosclerotic, human cadaver iliofemoral arteries. Ultrasonic energy caused dose (energy)-dependent relaxation in rabbit aortas after precontraction with phenylephrine in arteries with endothelium (n = 8) and without endothelium (n = 8) (p less than 0.001 versus ultrasound treated at power outputs of 2.9 and 5.5 W). There was no difference in the relaxation dose responses between endothelialized and endothelially denuded segments (p = NS). Ultrasonic energy also caused significant relaxation (67 +/- 8%) after voltage-dependent precontraction with 60 mM KCl. Temperature measurements revealed less than 1 degrees C warming of the vessel wall during as long as 2 minutes of treatment at a power output of 5.5 W. Pathological examination showed no smooth muscle injury at (moderate) power outputs that caused arterial relaxation. At probe-tip power outputs of 2.9-5.5 W, ultrasonic energy recanalized two of two totally occluded cadaveric iliofemoral vessel segments. The ultrasonic ablation catheter was also demonstrated to cause arterial relaxation in a recanalized canine femoral artery in vivo. CONCLUSIONS. Ultrasonic energy delivered via a flexible-wire probe produces dose-dependent, endothelium-independent smooth muscle relaxation capable of reversing both receptor-mediated and voltage-dependent vasoconstriction in vitro. At moderate power outputs, this relaxation response does not appear to be due to thermal effects or irreversible smooth muscle cell injury. This vasorelaxant effect of ultrasonic energy is also apparent in vivo, at doses that effectively ablate atherosclerotic plaque, and may improve the safety of arterial recanalization using ultrasonic energy.     

Acute induction of human IL-8 production by intestinal epithelium triggers neutrophil infiltration without mucosal injury

AIM: Neutrophil migration in the intestine depends on chemotaxis of neutrophils to CXC chemokines produced by epithelial cells. The goal of this project was to determine if acute induction of a CXC chemokine gradient originating from intestinal epithelial cells is sufficient to induce neutrophil influx into intact intestinal tissue. METHODS AND RESULTS: The authors developed a double transgenic mouse model with doxycycline induced human IL-8 expression restricted to intestinal epithelial cells. Doxycycline treatment of double transgenic mice for three days resulted in a 50-fold increase in the caecal IL-8 concentration and influx of neutrophils into the lamina propria. Although neutrophils entered the paracellular space between epithelial cells, complete transepithelial migration was not observed. Doxycycline treatment also increased the water content of the caecal and colonic stool, indicating dysfunctional water transport. However, the transmural electrical resistance was not decreased. Neutrophils recruited to the intestinal epithelium did not show evidence of degranulation and the epithelium remained intact as judged by histology. CONCLUSIONS: This conditional transgenic model of chemokine expression provides evidence that acute induction of IL-8 in the intestinal epithelium is sufficient to trigger neutrophil recruitment to the lamina propria, but additional activation signals are needed for full activation and degranulation of neutrophils, mucosal injury, and complete transepithelial migration.     

Impact of a multifaceted prevention program on ventilator-associated pneumonia including selective oropharyngeal decontamination

Purpose

We describe the impact of a multifaceted program for decreasing ventilator-associated pneumonia (VAP) after implementing nine preventive measures, including selective oropharyngeal decontamination (SOD).

Methods

We compared VAP rates during an 8-month pre-intervention period, a 12-month intervention period, and an 11-month post-intervention period in a cohort of patients who received mechanical ventilation (MV) for?>?48 h. The primary objective was to assess the effect on first VAP occurrence, using a Cox cause-specific proportional hazards model. Secondary objectives included the impact on emergence of antimicrobial resistance, antibiotic consumption, duration of MV, and ICU mortality.

Results

Pre-intervention, intervention and post-intervention VAP rates were 24.0, 11.0 and 3.9 VAP episodes per 1000 ventilation-days, respectively. VAP rates decreased by 56% [hazard ratio (HR) 0.44, 95% CI 0.29–0.65; P?<?0.001] in the intervention and by 85% (HR 0.15, 95% CI 0.08–0.27; P?<?0.001) in the post-intervention periods. During the intervention period, VAP rates decreased by 42% (HR 0.58, 95% CI 0.38–0.87; P?<?0.001) after implementation of eight preventive measures without SOD, and by 70% after adding SOD (HR 0.30, 95% CI 0.13–0.72; P?<?0.001) compared to the pre-intervention period. The incidence density of intrinsically resistant bacteria (to colistin or tobramycin) did not increase. We documented a significant reduction of days of therapy per 1000 patient-days of broad-spectrum antibiotic used to treat lower respiratory tract infection (P?<?0.028), median duration of MV (from 7.1 to 6.4 days; P?<?0.003) and ICU mortality (from 16.2 to 13.5%; P?<?0.049) for patients ventilated?>?48 h between the pre- and post-intervention periods.

Conclusions

Our preventive program produced a sustained decrease in VAP incidence. SOD provides an additive value.

     

Bilateral Orbital IgG4-Related Disease with Systemic and Corneal Involvement Showing an Excellent Response to Steroid and Rituximab Therapy: Report of a Case with 11 Years Follow-Up

IgG4-related disease is a newly recognized fibro-inflammatory condition. The purpose of this report is to present a patient with 11 years of follow-up, who revealed characteristic features of IgG4-related disease with systemic, orbital and corneal involvement and showed a favorable response to steroids and rituximab treatment.     

How marital relations affect child behavior: review of recent research

The development of children over time is one of the continuing aims of psychological inquiry. Many studies have concluded that children who grew up in dysfunctional households show inconsistent development across various developmental pathways. Previous studies have consistently concluded how different family factors, such as interparents relationship, parent–child relationships, and emotional relations in families, can explain the outcomes of child behavior problems. Therefore, this paper attempts to review the empirical findings of previous studies that assess the effects of marital relations on child behavioral development by reviewing the literature that supports the association between marital relations and behavioral problems of children. Twenty-seven studies were chosen for their outcomes. This review used online databases such as Google Scholar, Scopus, ProQuest, Psychological and Behavioral Science collection, SAGE full-text collection in psychology, and PsycINFO. The key words used were marital relation, marital conflict, marital satisfaction, child behavior problem, and offspring behavior adjustment. All articles hypothesized that marital relation affects child adjustment and behavior outcomes directly and indirectly. Approximately all the research samples were in the developmental stage (ages 3 to 12). Various methodologies were used in the studies, but some methodological defects in the literature in some way diminished the interpretation of their findings. Results revealed that children who come from families high in conflict are at risk of psychopathological developmental problems in their adolescent years. This review adds to a large collection of literature that highlights the damaging effects of marital conflict on children. Research limitations and suggestions for future studies are outlined. The study provides a concrete basis for further research on this topic considering that married couples and child behavior problems are varied. This review can benefit married and soon-to-be married couples by revealing perceptions into the experiences of others as regards marriage problem solving.