Detection of Salmonella spp. Using a Generic and Differential FRET-PCR

To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a T _m of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.     

Hydrophylita (Lutzimicron) emporos Shih & Polaszek (Hymenoptera: Trichogrammatidae) from Taiwan,Parasitising Eggs,and Phoretic on Adults,of the Damselfly Psolodesmus mandarinus mandarinus (Zygoptera: Calopterygidae)

Hydrophylita emporos n. sp. reared from eggs of Psolodesmus mandarinus mandarinus McLachlan (Zygoptera: Calopterygidae) in Taiwan is described. This is the first species of Hydrophylita to be described from the Old World, and the first record of phoresy in the genus. Adult females were observed aggregating at the base of the female damselfly’s abdomen. When the damselfly begins ovipositing, females move to the tip of the abdomen, enter the water and quickly locate eggs for parasitising. The article contains links to video footage of this process.     

Genetic Variants of LRRK2 in Taiwanese Parkinson’s Disease

Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson’s disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38–3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65–0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27–3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.     

The Post-Resuscitative Urinalysis Associate the Survival of Patients with Non-Traumatic Out-of-Hospital Cardiac Arrest

Objective

To analyze whether urine output and urinalysis results are predictive of survival and neurologic outcomes in patients with non-traumatic out-of-hospital cardiac arrest (OHCA).

Methods

Information was obtained from 1,340 patients with non-traumatic OHCA who had achieved a sustained return of spontaneous circulation (ROSC). Factors that were associated with survival in the post-resuscitative period were evaluated. The association between urine output and fluid challenge in the early resuscitative period was analyzed and compared between the survivors and the non-survivors. The results of the initial urinalysis, including the presence of proteinuria and other findings, were used to evaluate the severity of vascular protein leakage and survival. The association between proteinuria and the neurologic outcomes of the survivors was also analyzed. The clinical features of capillary leakage were examined during the post-resuscitative period.

Results

Of the 1,340 patients, 312 survived. A greater urine output was associated with a higher chance of survival. The initial urine output increased in proportion to the amount of fluid that was administered during early resuscitation in the emergency department for the survivors but not for the non-survivors (p<0.05). In the initial urinalysis, proteinuria was strongly associated with survival, and severe proteinuria indicated significantly poorer neurologic outcomes (p<0.05 for both comparisons). Proteinuria was associated with a risk of developing signs of capillary leakage, including body mass index gain and pitting edema (both p<0.001).

Conclusion

The severity of proteinuria during the early post-resuscitative period was predictive of survival.     

Re-Assessment of PrPSc Distribution in Sporadic and Variant CJD

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrP^Sc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrP^Sc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrP^Sc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrP^Sc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrP^Sc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrP^Sc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrP^Sc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrP^Sc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrP^Sc and infectivity.     

Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly,Ceratitis capitata

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.     

Efficacy of Sorafenib Monotherapy versus Sorafenib-Based Loco-Regional Treatments in Advanced Hepatocellular Carcinoma

Background

Although sorafenib is accepted as the standard of care in advanced hepatocellular carcinoma (HCC), its therapeutic benefit is marginal. Here, we aimed to compare the efficacy and safety of sorafenib monotherapy (S-M) and sorafenib-based loco-regional treatments (S-LRTs) in advanced HCC.

Methods

From 2007 to 2012, 290 patients with advanced HCC (Barcelona Clinic Liver Cancer stage C) with S-M (n = 226) or S-LRTs (n = 64) were reviewed retrospectively. Survival outcomes and treatment-related toxicities between two groups were analyzed.

Results

Variables related to tumor burden and liver function were similar between the groups (all P > 0.05). Within the entire population, the S-LRTs group had both longer median overall survival (OS) (8.5 vs 5.5 months, P = 0.001) and progression-free survival (PFS) (5.3 vs 3.0 months, P = 0.002) than the S-M group. Furthermore, the S-LRTs group had longer Os than the S-M group in a subgroup with neither extrahepatic spread (EHS) nor regional nodal involvement (RNI) (18.0 vs 7.8 months, P = 0.019) and in a subgroup with EHS and/or RNI (8.3 vs 4.8 months, P = 0.028). In addition, the S-LRTs group had longer PFS than the S-M group in the subgroup with neither EHS nor RNI (9.6 vs 3.2 months, P = 0.027).

Treatment

Related toxicity was similar between two groups.

Conclusion

Combined use of sorafenib and LRTs may provide better treatment outcomes without significantly increasing treatment-related toxicities, even in patients with EHS and/or RNI. Therefore, addition of active LRTs might be considered, if feasible.     

Celecoxib-Induced Cytotoxic Effect Is Potentiated by Inhibition of Autophagy in Human Urothelial Carcinoma Cells

Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, can elicit anti-tumor effects in various malignancies. Here, we sought to clarify the role of autophagy in celecoxib-induced cytotoxicity in human urothelial carcinoma (UC) cells. The results shows celecoxib induced cellular stress response such as endoplasmic reticulum (ER) stress, phosopho-SAPK/JNK, and phosopho-c-Jun as well as autophagosome formation in UC cells. Inhibition of autophagy by 3-methyladenine (3-MA), bafilomycin A1 or ATG7 knockdown potentiated celecoxib-induced apoptosis. Up-regulation of autophagy by rapamycin or GFP-LC3B-transfection alleviated celecoxib-induced cytotoxicity in UC cells. Taken together, the inhibition of autophagy enhances therapeutic efficacy of celecoxib in UC cells, suggesting a novel therapeutic strategy against UC.     

Knockdown of the Sodium-Dependent Phosphate Co-Transporter 2b (NPT2b) Suppresses Lung Tumorigenesis

The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras ^LA1 mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy.     

Augmenting Endogenous Wnt Signaling Improves Skin Wound Healing

Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2^LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2^LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2^LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice.