Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Shelterin reduces the accessibility of telomeric overhangs.

Telomeres terminate with a 50-300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction.

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24-144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3'- or 5'-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.

Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase.

G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2-3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

A single molecule investigation of i-motif stability, folding intermediates, and potential as in-situ pH sensor.

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Förster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.

Evaluation of normal or minimally elevated alanine transaminase, age and DNA level in predicting liver histological changes in chronic hepatitis B.

BACKGROUND: Serum alanine transaminase (ALT), hepatitis B virus (HBV) DNA level and age are used in the evaluation of chronic hepatitis B (CHB). AIM: We designed this study to evaluate liver histology with ALT and its relation with age and HBV DNA. METHODS: During the period of October 2006 to July 2009, 499 CHB patients were included in this study with detectable HBV DNA at PCR. Of these, 181 had normal ALT, 200 had ALT [>(1 × ULN) < (2 ULN)] and 118 had ALT ≥ 2 ULN and were labelled as Group 1, 2 and 3 respectively. RESULTS: A strong positive correlation was found between ALT and histological activity index (HAI) and fibrosis. However, 29 (52.7%) and five (9.1%) in Group 1 with positive HBeAg status had HAI ≥4 and fibrosis ≥2 respectively. Among those with HBeAg-negative status, 66 (23.1%) had HAI >4 and 31 (10.8%) had fibrosis ≥2. In Group 2, 14 (15.7%) had moderate-to-severe HAI and 19 (21.2%) had fibrosis ≥2 when HBeAg was positive, in those with HBeAg negative 34 (30.6%) had moderate-to-severe HAI and 38 (34.2%) had fibrosis ≥2. An ALT value of ≥58.5 U/l had higher sensitivity than that of 80 U/l in predicting significant histological changes. Further, HAI and fibrosis were significantly greater in the age of >30 years. CONCLUSIONS: We recommend liver biopsy in HBeAg-negative CHB over 30 years of age regardless of ALT level and starting treatment at ALT 1.5 × ULN instead of 2 × ULN.

Percutaneous liver biopsy: technique and safety.

BACKGROUND/AIMS: Percutaneous liver biopsy is a commonly used procedure for management of patients with liver diseases. We studied 107 patients of liver diseases with percutaneous liver biopsy to assess the need and usefulness of post procedure abdominal binder, analgesics, antibiotics or blood transfusion, and safety of the procedure. METHODOLOGY: We selected 107 consecutive patients having clear indication for liver biopsy. Each and every patient underwent percutaneous liver biopsy under uniform technique. The study was performed at the Department of Hepatology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh during the period from July 2006 to December 2007. RESULTS: Mean age of the patients was 27.35 years with +/- 7.62 (SD) years. Eighty five of them were male and 22 were female. No abdominal binder or antibiotic was used after the procedure. No analgesic or blood transfusion was required after the procedure. CONCLUSION: Routine post procedure use of abdominal binder and antibiotic are needless. Analgesics and blood transfusion are not always needed after the procedure. Percutaneous liver biopsy is a safe procedure in expert hands.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

Effect of Simultaneous Combined Treadmill Training and Magnetic Stimulation on Spasticity and Gait Impairments after Cervical Spinal Cord Injury.

Cervical spinal cord injury (CSCI) can induce lifelong disabilities, including spasticity and gait impairments. The objective of this pre-clinical study was to evaluate the therapeutic effects of simultaneous and combined early locomotor treadmill training (Tm) and injury site magnetic stimulation (TMSsc) on spasticity and gait impairments in a rat model of C6/7 moderate contusion SCI. The Tm training was initiated at post-injury (PI) day 8, whereas TMS treatment was added to Tm 14 days PI, and then the combined therapy (TMSTm) was continued for six weeks. Untreated CSCI animals revealed significant and enduring hindlimb spasticity (measured as velocity-dependent ankle torques and time-locked triceps surae electromyography), significant alterations in limb coordination, and significant reductions in forelimb grip strength. The TMSTm showed significantly lower spasticity, significantly more normal limb coordination (quantitated using three-dimensional (3D) kinematics and Catwalk gait analyses), and significantly greater forelimb grip strength compared with the CSCI untreated controls. In addition, three-dimensional gradient echo and diffusion tensor magnetic resonance imaging showed that TMSTm treated animals had smaller cavity volumes and better preservation of the white matter. In addition, compared with the CSCI untreated animals, the lumbar spinal cord (SC) of the treatment group revealed significant up-regulation of dopamine beta-hydroxylase, glutamic acid decarboxylase, gamma-aminobutyric acid receptor B, and brain-derived neurotrophic factor. The treatment-induced up-regulation of these molecules may have enhanced the activity-induced adaptive plasticity in the SC and contributed to normalization of pre- and post-synaptic reflex regulatory processes. In addition, the TMSTm therapy may have decreased injury-induced progressive maladaptive segmental and descending plasticity. Our data are the first to suggest that an early simultaneous combination of Tm and injury-site TMSsc application can be an effective therapy for CSCI-induced spasticity and gait impairments. These pre-clinical data demonstrated the feasibility and efficacy of a novel therapeutic strategy for SCI-induced spasticity and gait impairments.

Clinical, anthropometric, biochemical and histological character of nonalcoholic fatty liver disease without Insulin Resistance.

OBJECTIVES: Nonalcoholic Fatty Liver Disease (NAFLD) is thought to be a hepatic manifestation of Metabolic Syndrome (MS) or Insulin Resistance (IR). The aim of the study was to explore the clinical, anthropometric, metabolic, biochemical and histological profile of NAFLD patients without IR by comparing it with NAFLD with IR. METHODS: Total 851 patients with sonographic evidence of fatty liver were included. These patients underwent clinical, anthropometric, biochemical and histological evaluation. IR was calculated using the homeostatic model assessment. Liver biopsy done in 285 patients who consented for the procedure and who had MS or raised ALT. RESULTS: Among 851 NAFLD patients, 561(65.9%) patients were without IR and 290 (34.1%) patients were with IR. The proportion of male sex [230 (41.0%) vs. 89 (30.7%); P = 0.046] were higher but diabetes [19.10% vs. 39.0%; P = 0.000] and MS were [58.80%vs. 78.10%; P = 0.014] significantly lower in non IR group. Body Mass Index (BMI) kg/m2 and Waist Circumference (WC) in cm were also lower in non IR group: [26.6 ± 3.5 vs. 27.9 ± 4.3; P = 0.002] and [93.3 ± 8.4 vs. 95.9 ± 8.4; P = .003]. Lipid profile, ALT, AST and ALP were not differed between the groups. Histopathology reports revealed that lobular inflammation, ballooning and fibrosis were similar in two groups, only steatosis score was higher in IR group [2.0 ± 0.7 vs. 1.8 ± 0.8; P = 0.007]. CONCLUSION: There are significant proportion of NAFLD patients without IR in Bangladesh. NAFLD patients without IR predominantly male, had lower BMI, WC, MS and diabetes. Histologically NAFLD without IR equally severe with ballooning, lobular inflammation and fibrosis except steatosis. Insulin resistance is the principal but not the sole factor for NAFLD in our population.

Function of a bound ubiquinone in Escherichia coli quinoprotein glucose dehydrogenase.

Membrane-bound glucose dehydrogenase (mGDH) is a single integral protein in the respiratory chain in Escherichia coli which oxidizes D-glucose and feeds electrons to ubiquinol oxidase via bulk ubiquinone (UQ). mGDH contains a bound UQ, CoQ8, for its intramolecular electron transfer in addition to pyrroloquinoline quinone (PQQ) as a coenzyme. Pulse radiolysis analysis revealed that the bound UQ exists very close to PQQ at a distance of 11-13 angstroms. Studies on mGDH mutants with substitutions for amino acid residues around PQQ showed that Asp-466 and Lys-493, which are crucial for catalytic activity, interact with bound UQ. Based on these findings, we propose that the bound UQ is involved in the catalytic reaction in addition to the intramolecular electron transfer in mGDH.

e-antigen-negative chronic hepatitis B in Bangladesh.

BACKGROUND: Bangladesh is a densely populated country where about 10 million people are chronically infected with hepatitis B virus (HBV). The aim of the present study was to evaluate the biochemical, virological and histological characteristics of HBeAg-negative chronic hepatitis B (CHB). METHODS: Patients were included in this study if they were chronically infected with HBV with detectable DNA. The patients who were co-infected with human immunodeficiency virus, hepatitis delta virus or hepatitis C virus, and previously subjected to antiviral treatment, and those with hepatocellular carcinoma were excluded. The study was conducted during the period of January 2001 to December 2007. During this period 2617 patients with CHB were studied. HBeAg-positive cases were included to compare the characteristics. Among them, 237 cases underwent liver biopsy. RESULTS: 2296 patients (87.7%) were male, with a mean age of 28.9+/-13.7 years. 2375 patients (90.8%) had CHB, and 242 (9.2%) were cirrhotic. HBV DNA levels were 7.6+/-1.5 copies/ml, ALT was 111.3+/-212.5 U/L, and AST was 91.5+/-148.9 U/L. The number of HBeAg-negative CHB cases was 1039 (39.7%). HBeAg-negative patients with a lower DNA load were older, and they had more fibrotic changes in the liver than HBeAg-positive patients. The two groups did not differ in necroinflammatory activity, but the former had lower ALT and AST values. Cirrhosis was more common in e-antigen-negative patients. CONCLUSIONS: e-antigen-negative CHB patients are older and have more hepatic fibrosis patients than HBeAg-positive patients, although they have similar necroinflammatory activity.

Effect of sitagliptin on hepatic histological activity and fibrosis of nonalcoholic steatohepatitis patients: a 1-year randomized control trial.

BACKGROUND/PURPOSE: Dipeptidyl peptidase 4 (DPP-4) expression is directly associated with hepatic lipogenesis and liver injury in nonalcoholic steatohepatitis (NASH). This study has been designed to elucidate the histological improvement of NASH with the DPP-4 inhibitor sitagliptin. MATERIALS AND METHODS: In this open-label randomized control trial, paired liver biopsy was taken from 40 NASH patients. Sitagliptin 100 mg was given once daily to the SL group and no sitagliptin was given to the L group for 1 year. Patients from both groups were encouraged to exercise moderately and advised to avoid saturated fat, excessive sugar, soft drinks, fast food, and refined carbohydrates to reduce weight. RESULTS: Steatosis improved in the SL group (from 2.3±0.6 to 1.2±0.8; P=0.000) and the L group (from 2.1±0.6 to 1.6±0.9; P=0.008), ballooning decreased from 1.8±0.6 to 1.3±06 (P=0.002) in the SL group, but not in the L group. Nonalcoholic fatty liver disease activity score (NAS) attenuated in both groups: the SL group (from 5.8±0.9 to 3.9±1.4; P=0.000) and the L group (from 5.3±0.6 to 4.6±1.2; P=0.009). NAS improvement was much higher in the SL group (1.9±1.4) than in the L group (0.7±1.1) (P=0.006), with NAS improving by ≥2 in 13 patients from the SL group and five patients from the L group (P=0.01). Improvement was irrespective of diabetes. Regression analysis explored that sitagliptin had odds of 6.38 and weight reduction had odds of 4.51 for NAS reduction. CONCLUSION: Sitagliptin 100 mg once daily for 1 year ameliorates NAS by improving steatosis and ballooning, irrespective of diabetes. Sitagliptin has stronger efficacy than that of weight reduction.

A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA.

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

Prevalence and risk factors of non-alcoholic fatty liver disease in Bangladesh.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) is a significant cause of hepatic dysfunction and liver-related mortality. As there is a lack of population-based prevalence data in a representative sample of general population, we aimed to estimate the prevalence and risk factors of NAFLD in Bangladesh. METHODS: A cross-sectional study was conducted both in urban and rural areas of Bangladesh from December 2015 to January 2017. Data were collected using a pretested structured questionnaire followed by ultrasonography of hepatobiliary system for screening of NAFLD. Multivariate logistic regression was used to estimate the risk factors of NAFLD. RESULTS: A total of 2782 (1694 men and 1088 women) participants were included in the study, with a mean age of 34.21 (±12.66) years. The overall prevalence of NAFLD was 33.86% (95% confidence interval [CI]: 32.12, 35.64). Females living in the rural areas and midlife adults (45-54 years) had the highest prevalence of NAFLD (P < 0.05). Multivariable logistic regression model demonstrated that increasing age, diabetes, elevated body mass index, and married individuals are significantly associated with NAFLD. Individuals with diabetes (adjusted odds ratio: 2.71, 95% CI: 1.85, 3.97) and hypertension were at a higher risk of having NAFLD. The odds of having NAFLD were 4.51 (95% CI: 3.47, 5.86) and 10.71 (95% CI: 7.80, 14.70) times higher among overweight and obese participants, respectively, as compared to normal-weight participants. CONCLUSIONS: About one-third of the population of Bangladesh is affected by NAFLD. Individuals with higher body mass index (overweight and obese), diabetics, midlife adults, married individuals, and rural women were more at risk of having NAFLD than others.

Study on ascitic fluid complement 3 level in cirrhotic patients with spontaneous bacterial peritonitis and without spontaneous bacterial peritonitis.

BACKGROUND/AIMS: Ascitic fluid Complement 3 (C3) concentration is the most important factor to offer local defense against infection of ascitic fluid. Hepatic synthesis of Complement 3 and its concentration in ascitic fluid is significantly reduced in patients with advanced cirrhosis. The aim of the study was to assess the level of Complement 3 in ascitic fluid in cirrhotic patients with and without spontaneous bacterial peritonitis (SBP) and to identify the group of cirrhotic ascites at risk of developing METHODOLOGY: A prospective case control study was carried out to compare the level of ascitic fluid Complement 3 concentration in patients with SBP (case-group) and without SBP (control-group). Ascitic fluid Complement 3 level was estimated in 15 patients with SBP (case) and another 15 patients without SBP (control). RESULTS: In the study, ascitic fluid Complement 3 concentration was 7.3+/-4.3 mg/dL in patients with SBP and 16.4+/-11.3 mg/dL in patients who did not develop SBP. CONCLUSIONS: Ascitic fluid Complement 3 level is significantly (P=0.009) reduced in cirrhotic patients who develop SBP.

Presentation and endoscopic management of biliary ascariasis.

Ascaris lumbricoides is a common parasite and the most serious and dramatic presentation is hepatobiliary and pancreatic ascariasis (HPA). Therefore, this study was planned prospectively to elucidate the clinical presentation of HPA and evaluate the efficacy and safety of endoscopic intervention. In this study we documented 77 consecutive patients with HPA from January 2000 to November 2005. All the patients had endoscopically proven HPA. A total of 77 patients were included in the study. The age ranged from 6 to 80 years, with the third decade most commonly (28.6%) affected. Females were 6 times more likely to be affected than males. The commonest presentation was biliary colic (97.4%); other presentations were acute cholangitis (15.6%), obstructive jaundice (9.1%), acute pancreatitis (6.5%), choledocholithiasis (6.5%), acute cholecystitis (6.5%) and liver abscess (2.6%). In this report 51 (66.2%) had living, 10 (13%) had dead and 16 (20.8%) had both living and dead worms. Choledocholithiasis was associated only with dead worms. From one to 23 worms were found in the biliary tree. In 94.8% of cases we had to remove the worm by wide papillotomy followed by basket extraction. We did not experience any major complications during or following the procedures. Three patients had recurrent HPA during the course of follow-up (1 to 12 months). The majority of patients with HPA presented with biliary colic. This should be kept in mind in the management of an acute abdomen, especially in tropical countries. Endoscopic extraction is a safe and effective procedure for the treatment of HPA.

Effect of Pentoxifylline on Histological Activity and Fibrosis of Nonalcoholic Steatohepatitis Patients: A One Year Randomized Control Trial.

BACKGROUND AND OBJECTIVES: To observe the effect of Pentoxifylline for 1 year on hepatic histological activity and fibrosis of nonalcoholic steatohepatitis (NASH). MATERIALS AND METHODS: A single center, open label Randomized Control Trial. Patients were included if they had ultrasonographic evidence of fatty liver and nonalcoholic fatty liver disease activity score (NAS) ≥ 5 on liver histology. A total of 35 patients were selected; 25 of PL (Experimental) group and 10 of L (Control) group. PL group received 400 mg pentoxifylline thrice daily along with lifestyle modification and there was only lifestyle modification for the L group. After one year, NAS and fibrosis was compared in both groups. RESULTS: In PL group, NAS improved 2.10 ± 1.07; whereas in L group, NAS was 0.90 ± 0.99 (P = 0.006). As per the protocol analysis, NAS ≥ 2 improved in 15/20 (75%) in PL group and in 3/10 (30%) in L group (P = 0.018). In PL group, the individual component of NAS, steatosis improved from 2.30 ± 0.66 to 0.95 ± 0.76 (P = 0.000), lobular inflammation from 1.65 ± 0.59 to 1.05 ± 0.51 (P = 0.002) and hepatocyte ballooning from 1.50 ± 0.51 to 1.30 ± 0.57 (P = 0.258). In L group, steatosis improved from 2.30 ± 0.68 to 1.40 ± 1.08 (P = 0.01), lobular inflammation and hepatocyte ballooning did not improve. The fibrosis score did not improve in any group. In PL group, NAS improved significantly (P = 0.027; OR=22.76, CI=1.43-362.40) independent of weight reduction. CONCLUSION: Pentoxifylline for 1 year improves the hepatic histological activity but not fibrosis of NASH patients.

Mild and Mild-to-Moderate Traumatic Brain Injury-Induced Significant Progressive and Enduring Multiple Comorbidities.

Traumatic brain injury (TBI) can produce life-long disabilities, including anxiety, cognitive, balance, and motor deficits. The experimental model of closed head TBI (cTBI) induced by weight drop/impact acceleration is known to produce hallmark TBI injuries. However, comprehensive long-term characterization of comorbidities induced by graded mild-to- mild/moderate intensities using this experimental cTBI model has not been reported. The present study used two intensities of weight drop (1.0 m and 1.25 m/450 g) to produce cTBI in a rat model to investigate initial and long-term disability of four comorbidities: anxiety, cognitive, vestibulomotor, and spinal reflex that related to spasticity. TBI and sham injuries were produced under general anesthesia. Time for righting recoveries post-TBI recorded to estimate duration of unconsciousness, revealed that the TBI mild/moderate group required a mean of 1 min 27 sec longer than the values observed for noninjured sham animals. Screening magnetic resonance imaging images revealed no anatomical changes, mid-line shifts, or hemorrhagic volumes. However, compared to sham injuries, significant long-term anxiety, cognitive, balance, and physiological changes in motor reflex related to spasticity were observed post-TBI for both TBI intensities. The longitudinal trajectory of anxiety and balance disabilities tested at 2, 4, 8, and 18 weeks revealed progressively worsening disabilities. In general, disability magnitudes were proportional to injury intensity for three of the four measures. A natural hypothesis would pose that all disabilities would increase incrementally relative to injury severity. Surprisingly, anxiety disability progressed over time to be greater in the mildest injury. Collectively, translational implications of these observations suggest that patients with mild TBI should be evaluated longitudinally at multiple time points, and that anxiety disorder could potentially have a particularly low threshold for appearance and progressively worsen post-injury.

Association of single nucleotide polymorphism at PNPLA3 with fatty liver, steatohepatitis, and cirrhosis of liver.

BACKGROUND/PURPOSE: The aim of this study was to determine the association of single nucleotide polymorphism (SNP) in patatin-like phospholipase domain-containing 3 (PNPLA3) at I148 with histological severity of non-alcoholic fatty liver disease (NAFLD). METHODS: Patients were selected for the study if they had histological evidence of NAFLD and clinical evidence of non-alcoholic steatohepatits (NASH) cirrhosis. We included 50 NASH cirrhosis, 99 patients of NAFLD including 36 non-NASH fatty liver (NNFL) along with 63 NASH and 75 healthy controls. PNPLA3 genotyping was done by real-time PCR using a Taqman assay for rs738409. RESULTS: CC, CG, and GG frequencies were 45 (60.0%)/27 (36.0%)/3 (4.0%) in healthy control, 19 (52.8%)/14 (38.9%)/ 3 (8.3%) in NNFL, 18 (28.6%)/29 (46.0%)/16 (25.4%) in NASH, and 7 (14.6%), 25 (52.1%), 16 (33.3%) in cirrhosis. The frequency of G allele was significantly higher (62.6%) in NAFLD than in healthy control. The GG genotype had 20.25 times odds of NAFLD. The GG genotype had 6.53 times odds of having NASH. HOMA-IR > 1.6 had 3.81 times odds of having NASH. Regression analysis revealed that G allele odds of having cirrhosis was 3.9 times compared to C. The G allele was also significantly associated with steatosis, lobular inflammation, NAFLD activity score, and fibrosis. CONCLUSION: PNPLA3 genotype showed an association with NAFLD, NASH, fibrosis, and cirrhosis.