New frontiers in the treatment of colorectal cancer: Autophagy and the unfolded protein response as promising targets.

Colorectal cancer (CRC), despite numerous therapeutic and screening attempts, still remains a major life-threatening malignancy. CRC etiology entails both genetic and environmental factors. Macroautophagy/autophagy and the unfolded protein response (UPR) are fundamental mechanisms involved in the regulation of cellular responses to environmental and genetic stresses. Both pathways are interconnected and regulate cellular responses to apoptotic stimuli. In this review, we address the epidemiology and risk factors of CRC, including genetic mutations leading to the occurrence of the disease. Next, we discuss mutations of genes related to autophagy and the UPR in CRC. Then, we discuss how autophagy and the UPR are involved in the regulation of CRC and how they associate with obesity and inflammatory responses in CRC. Finally, we provide perspectives for the modulation of autophagy and the UPR as new therapeutic options for CRC treatment.

[Characteristics of main layer and regeneration layer of Haloxylon ammodendron plantations at different ages on the southern edge of the Gurbantunggut Desert, Northwest China]. / å¤å°”ç­é€šå¤ç‰¹æ²™æ¼ å—ç¼˜ä¸åŒæž—é¾„äººå·¥æ¢­æ¢­æž—ä¸»æž—å±‚å’Œæ›´æ–°å±‚ç‰¹å¾.

The population structure characteristics, natural regeneration, and the influential factors of Haloxylon ammodendron plantations at six different stand ages on the southern edge of the Gurbantunggut Desert were studied. The results showed that H. ammodendron plantation at the stand age of 7 could naturally regenerate. At the stand age of 17, the densities of the seedlings (＜30 cm height), saplings (30≤H＜50 height), and small trees (≥50 cm height) reached optimal class, and the mean height and base diameter of the small tress reached 1.10 m and 1.91 cm, respectively. The parent trees in H. ammodendron plantation at the stand age of 20 grew best. The height of 35% individuals grew up to 2.50-3.00 m, and the basal stem diameter of 23.1% individuals grew up to 8.00-10.00 cm. The height and diameter growth of the parent trees in H. ammodendron plantation at the stand age of 33 apparently declined, but the regeneration ability by natural seed dispersal was still strong. The regeneration density of natural seed dispersal showed the greatest correlation with the available nitrogen content in 0-100 cm soil layer (0.87), followed by the soil rapidly available phosphorus content (0.84) and the soil water content (0.79). The soils with pH 8.1-8.6 did not limit the nutrient growth of the regeneration layer. In the main stand layer, the individual density of whole regeneration layer showed the greatest correlation with the biomass of the parent trees (0.77), while the density of regeneration layer of the small trees showed the greatest correlation with the planting density (0.71) and the age of the parent trees (0.70).

Mevalonate Cascade and Neurodevelopmental and Neurodegenerative Diseases: Future Targets for Therapeutic Application.

The mevalonate cascade is a key metabolic pathway that regulates a variety of cellular functions and is thereby implicated in the pathophysiology of most brain diseases, including neurodevelopmental and neurodegenerative disorders. Emerging lines of evidence suggest that statins and Rho GTPase inhibitors are efficacious and have advantageous properties in treatment of different pathologic conditions that are relevant to the central nervous system. Beyond the original role of statins in lowering cholesterol synthesis, they have anti-inflammatory, antioxidant and modulatory effects on signaling pathways. Additionally, Rho GTPase inhibitors and statins share the mevalonate pathway as a common target of their therapeutic actions. In this review, we discuss potential mechanisms through which these drugs, via their role in the mevalonate pathway, exert their neuroprotective effects in neurodegenerative and neurodevelopmental disorders.

Mevalonate Cascade and Small Rho GTPase in Spinal Cord Injury.

The mevalonate pathway has been extensively studied for its involvement in cholesterol synthesis. Inhibition of this pathway using statins (3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors; HMGR inhibitors) is the primarily selected method due to its cholesterol-lowering effect, making statins the most commonly used (86-94%) cholesterol-lowering drugs in adults. This pathway has several other by-products that are affected by statins including GTPase molecules (guanine triphosphate-binding kinases), such as Rho/Rho-associated coiled kinase (ROCK) kinases, that are implicated in other diseases, including those of the central nervous system (CNS). These molecules control several aspects of neural cell life including axonal growth, cellular migration, and cell death, and therefore, are of increasing interest in the field of spinal cord injury (SCI). Limited regeneration capacity of nerve fibers in adult CNS has been considered the main obstacle for finding a SCI cure. Over the past two decades, the identity of inhibitory factors for regeneration has been widely investigated. It is well-established that the Rho/ROCK kinase system is specifically activated by the components of damaged spinal cord tissue, including oligodendrocytes and myelin, as well as extracellular matrix. This has led many groups to hypothesize that statin therapy may in fact enhance the current neurorestorative approaches. In this mini-review, a summary of SCI pathophysiology is discussed and the current literature targeting the regeneration obstacles in SCI are reviewed, with special attention to recent publications of the past decade. In addition, we focus on the current literature involving the use of pharmacological and molecular inhibitors of small GTPase molecules for treatment of neurotrauma. Inhibiting these molecules has been shown to increase neuroprotection, enhance axonal regeneration, and facilitate the implementation of cell replacement therapies. Based upon available literature, the need for clinical trials involving targeted inhibition of GTPase molecules remains strong. Some of these drugs are widely used for other diseases, and therefore re-purposing their application for neurotrauma can be fasttracked. These approaches can potentially modify the inhibitory environment of nervous tissue to allow the spontaneous repair capacity of injured tissue.

Liver transplantation for biliary atresia: A single-center study from mainland China.

AIM: To summarize our single-center experience with liver transplantation (LT) for biliary atresia (BA). METHODS: From October 2006 to December 2012, 188 children with BA were analyzed retrospectively. The stage I group (from October 2006 to December 2010) comprised the first 74 patients, and the stage II group (from January 2011 to December 2012) comprised the remaining 114 patients. Finally, 123 liver transplants were performed in 122 (64.9%) patients, whereas 66 patients did not undergo LT due to denial by their parents or lack of suitable liver grafts. The selection of graft types depended on the patients' clinical status and whether a suitable living donor was available. The characteristics of patients in stages I and II were described, and the surgical outcomes of LT recipients were compared between the two stages. The Kaplan-Meier method was used to estimate the cumulative patient and graft survival rates, and the equality of survival distributions was evaluated using the log-rank test. RESULTS: The 188 children consisted of 102 boys and 86 girls. Their ages ranged from 3 to 144 mo with a median of 8 mo. One hundred and fifteen (61.2%) patients were born in rural areas. Comparing stage I and stage II patients, the proportion of patients referred by pediatricians (43.2% vs 71.1%, respectively; P < 0.001) and the proportion of patients who previously received a Kasai procedure (KP) (32.4% vs 44.7%, respectively; P = 0.092) obviously increased, and significantly more parents were willing to treat their children with LT (73% vs 86%, respectively; P = 0.027). Grafts from living donors (102/122, 83.6%) were the most commonly used graft type. Surgical complications (16/25, 64.0%) were the main reason for posttransplant mortality. Among the living donor liver transplantation recipients (n = 102), the incidence of surgical complications was significantly reduced (34.1% vs 15.5%, respectively; P = 0.029) and survival rates of patients and grafts were greatly improved (81.8% vs 89.7%, respectively, at 1 year; 75.0% vs 87.8%, respectively, at 3 years; P = 0.107) from stage I to stage II. CONCLUSION: The status of surgical treatments for BA has been changing in mainland China. Favorable midterm outcomes after LT were achieved as centers gained greater technical experience.

[Spatial distribution pattern of main populations and gap makers in Picea koraiensis and Abies nephrolepis forest of Xiaoxing' an Mountains, Northeast China].

Species composition and diameter class structure were investigated in 1.5 hm2 (100 m x 150 n) permanent plot in Picea koraiensis and Abies nephrolepis forest of Xiaoxing' an Mountains. The spatial distribution pattern and spatial association of main populations and gap makers were analyzed by using point pattern analysis. The results showed that there were a total of 13 species with diameters at breast height greater than 2 cm in tree layer, and great differences were observed in the densities of main populations. The importance values of A. nephrolepis, P. koraiensis, Betula platyphylla and Acer ukurunduense were ranked in the first 4 in the plot. The diameter class structure of their populations presented an inverse 'J' curve. The spatial distribution patterns for A. nephrolepis and P. koraiensis were similar, which changed from aggregated, random to uniform distribution with the spatial scale. For B. platyphylla, the distribution was aggregated at ≤40 m scale, and random at >40 m scale, whereas A. ukurunduense presented an aggregated distribution pattern at the whole research scale. Except that the negative correlation between B. platyphylla and A. ukurunduense existed at the whole research scale, positive correlation between the other populations at small scale and negative correlation at large scale were observed. Only A. nephrolepis and B. platyphylla had significant positive correlation, and generally no significant correlation existed between other populations. Spatial distribution pattern of gap makers was characterized as aggregated distribution at small and middle scales, and random distribution with increasing scale. Spatial point pattern of gap makers formed by uprooting exhibited unimodal type distribution, and random, aggregated, and uniform distribution also occurred. Spatial point pattern of gap makers formed by breaking overall presented a little fluctuation, random and aggregated distributions alternatively appeared at small scale, and random distribution happened at large scale. Their spatial correlation analysis indicated that significant positive correlation existed at ≤32 m scale, and non-significant negative correlation at >32 m scale.

Bovine HEXIM1 inhibits bovine immunodeficiency virus replication through regulating BTat-mediated transactivation.

The bovine immunodeficiency virus (BIV) transactivator (BTat) recruits the bovine cyclin T1 (B-cyclin T1) to the LTR to facilitate the transcription of BIV. Here, we demonstrate that bovine hexamethylene bisacetamide (HMBA)-induced protein 1 (BHEXIM1) inhibits BTat-mediated BIV LTR transcription. The results of in vivo and in vitro assays show direct binding of BHEXIM1 to the B-cyclin T1. These results suggest that the repression arises from BHEXIM1-BTat competition for B-cyclin T1, which allows BHEXIM1 to displace BTat from B-cyclin T1. Furthermore, we found that the C-terminal region and the centrally located region of BHEXIM1 are required for BHEXIM1 to associate with B-cyclin T1. Knockdown of BHEXIM1 enhances BIV replication. Taken together, our study provides the first clear evidence that BHEXIM1 is involved in BIV replication through regulating BTat-mediated transactivation.

A new indicator cell line established to monitor bovine foamy virus infection.

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

[Effect of protocol RS-99 for childhood rhabdomyosarcoma].

OBJECTIVE: To develop a reasonable protocol for childhood rhabdomyosarcoma (RS) to improve the prognosis of this disease. METHODS: The protocol RS-99, developed on the base of pathological examination, clinical staging and grouping was used for 30 RS patients, 15 male and 15 female, aged 53 months (15-180 months). For the low-risk patients the regimen VCP and IeV were used alternately for 6 treatment courses and then local radiotherapy was given, for the median and high risk patients, regimen AVCP and IEV were used alternately for 6 courses, local radiotherapy and selective operation were performed, and then DEV and IeV were used alternately for 6 courses, and for the high-risk patients DEV and IeV were used alternately for 18 courses in total and then hematopoietic stem cell transplantation was conducted. RESULTS: Twenty-three of the 30 patients reached complete response (CR) 17 of which remained in CR for 37 (32-92) months, and 7 of the 30 patients achieved partial response (PR) but their condition worsened later. Relapse was seen in 6 patients 15 (7-38) months later. The 5 year event-free survival (EFS) rate was 47% and the overall survival (OS) rate was 52%. All the 10 stage I and II patients, 5 of the 8 stage III patients, and only 2 of the 12 stage IV patients still remained in CR. CONCLUSION: The protocol RS-99 is effective on the RS at stages I, II, and III, however, is ineffective on the disease at stage IV.

Cloning and characterization of a novel intracellular protein p48.2 that negatively regulates cell cycle progression.

Neurofibromatosis type 1 (NF1) microdeletion is a large genomic deletion that embraces at least 11 continuous genes at human chromosome 17q11.2. To date, most of these genes' functions still remain undefined. In this study, we report an unknown cytokine receptor like molecule (p48.2) that is frequently deleted in patients with type-1 and type-2 NF1 microdeletions in the neurofibromin locus. The cloned gene has 1317 base pair long that encodes a 438aa intracellular protein. The gene was subsequently named p48.2 based on its predicted molecular weight. A typical fibronectin type III (FNIII) domain was identified in p48.2 between Arg(176) and Pro(261) in which a palindromic Arg-Gly-Asp (RGD) repeat plus a putative Trp-Ser-X-Trp-Ser (WSXWS) motif were found at the domain's C-terminus. p48.2 mRNAs were abundant in many tumor cell lines and normal human tissues and up-regulated in some freshly isolated lung cancer and leukemia cells. Interestingly, over-expression of p48.2 in human embryo kidney 293T cells could significantly cause G0/G1 arrest and prevented S phase entry. In contrast, repressing endogenous p48.2 gene expression by specific siRNA markedly reduced G0/G1 population. Importantly, over-expression of p48.2 could significantly up-regulate rather than down-regulate cyclin D1 and cyclin D3 expressions. We further showed that the induction of cyclin D1 expression was directly due to the activation of signal transducers and activators of transcription 3 (STAT3), but was independent of RAS/mitogen-activated protein kinase (RAS/MAPK) signaling pathway. Thus, p48.2 may represent a novel type of intracellular protein functioning as a negative regulator at the G0/G1 phase.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain.

BACKGROUND: The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07_BC. A highly replication-competent simian/human immunodeficiency viruses (SHIV) construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell (PBMC), establish infection in Chinese rhesus macaque. METHODS: A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques (Macaca mulatta) was infected with one SHIV via intravenous infusion. RESULTS: One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. CONCLUSIONS: We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF_07BC recombinant strain.

Detection and analysis of bovine foamy virus infection by an indicator cell line.

AIM: To determine the infectivity and replication strategy of bovine foamy virus (BFV) in different cultured cells using the BFV indicator cell line (BICL) system. METHODS: BFV infection was induced by the co-culture method or the transient transfection of the infectious BFV plasmid [pCMV (cytomegalovirus) - BFV] clone. The infectivity of BFV was monitored by the percentage of green fluorescent protein-positive cells in the BICL. The effect of reverse transcriptase inhibitor zidovudine (AZT) on BFV replication was also evaluated in the BICL. RESULTS: The titer of BFV in fetal bovine lung cells was 4-5-folds more than that in either 293T or HeLa (Cells from Henrietta lacks) cells using the co-culture method, and in the meantime was significantly higher than that produced by the infectious clone pCMV-BFV in the same cells. AZT had only a minor effect on viral titers when added to cells prior to the virus infection. In contrast, viral titers reduced sharply to the level of the negative control when the virus was produced from cells in the presence of AZT. CONCLUSIONS: BICL can be used for the titration of the BFV viral infection in non-cytopathic condition. In addition, we provide important evidence to show that reverse transcription is essential for BFV replication at a late step of viral infection.

Tight filum terminale syndrome in children: analysis based on positioning of the conus and absence or presence of lumbosacral lipoma.

BACKGROUND: Tight filum terminale syndrome (TFTS) characterized by findings consistent with a tethered cord but with the conus ending in a normal position has only recently been observed in children. In this situation, diagnosis may prove difficult and sectioning of the filum terminale is questionable. MATERIALS AND METHODS: Sixty cases of pediatric TFTS were analyzed by methods including spinal X-ray and magnetic resonance imaging (MRI). Twenty-one patients exhibited a normally positioned conus, 18 a low-lying conus, and 21 a low-lying conus with accompanying lumbosacral lipoma. These three groups were compared preoperatively and postoperatively for lumbosacral cutaneous stigmata, vertebral anomalies, concomitant congenital spinal dysraphisms, lower limb deformities, and sphincter dysfunction. RESULTS: Rates of occurrence of lumbosacral cutaneous stigmata and other concomitant congenital spinal dysraphisms differed significantly among the groups. Differences in other parameters were not observed. All groups responded positively to surgery. CONCLUSIONS: Pediatric TFTS may involve a normally positioned conus. Diagnosis of pediatric TFTS should be based on clinical presentation, physical and radiological examinations, MRI, and pathologic changes in the filum. When neurological signs accompany such changes, early severing of the filum is indicated regardless of conus position.

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene.

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses. METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supernatant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells. RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells. CONCLUSION: The replacement of partial gag gene of HIV with BIV gag gene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

[Construction and analysis of activity of an HIV-1/bovine immunodeficiency virus chimeric clone cDNA].

OBJECTIVE: Chimeric human/bovine immunodeficiency virus (HBIV) cDNA was constructed by replacing HIV tat and LTR with bovine immunodeficiency virus (BIV) tat and LTR to study the activity of BIV tat and LTR in the chimerae. METHODS: The target fragments of BIV tat, LTR and HIV gag, pol, env were respectively amplified by using PCR and sequentially inserted into pBluescript SK(+) vector. The chimeric clone was transfected into human MT4 cells. The transcript and gene expression of the HBIV chimeric virus were detected by using RT-PCR and a reverse transcriptase assay, respectively. RESULTS: BIV tat mRNA and HIV gag mRNA were detected. The reverse transcriptase activity of the chimeric virus was analyzed in the fluctuation curve. CONCLUSIONS: In chimeric HBIV cDNA transfected MT?4 cells, BIV tat and HIV gag were transcripted. The reverse transcriptase of the chimeric virus had biological activity. These data suggest that in MT4 cells, BIV LTR had promoter activity and BIV tat had the function of transactivation in the chimeric virus. The study of the chimeric virus with infectivity is in progress.