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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mild closed head traumatic brain injury-induced changes in monoamine neurotransmitters in the trigeminal subnuclei of a rat model: mechanisms underlying orofacial allodynias and headache.

Our recent findings have demonstrated that rodent models of closed head traumatic brain injury exhibit comprehensive evidence of progressive and enduring orofacial allodynias, a hypersensitive pain response induced by non-painful stimulation. These allodynias, tested using thermal hyperalgesia, correlated with changes in several known pain signaling receptors and molecules along the trigeminal pain pathway, especially in the trigeminal nucleus caudalis. This study focused to extend our previous work to investigate the changes in monoamine neurotransmitter immunoreactivity changes in spinal trigeminal nucleus oralis, pars interpolaris and nucleus tractus solitaries following mild to moderate closed head traumatic brain injury, which are related to tactile allodynia, touch-pressure sensitivity, and visceral pain. Our results exhibited significant alterations in the excitatory monoamine, serotonin, in spinal trigeminal nucleus oralis and pars interpolaris which usually modulate tactile and mechanical sensitivity in addition to the thermal sensitivity. Moreover, we also detected a robust alteration in the expression of serotonin, and inhibitory molecule norepinephrine in the nucleus tractus solitaries, which might indicate the possibility of an alteration in visceral pain, and existence of other morbidities related to solitary nucleus dysfunction in this rodent model of mild to moderate closed head traumatic brain injury. Collectively, widespread changes in monoamine neurotransmitter may be related to orofacial allodynhias and headache after traumatic brain injury.

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.

Prolonged hippocampal cell death following closed-head traumatic brain injury in rats.

Traumatic brain injury (TBI) leads to enduring cognitive disorders. Although recent evidence has shown that controlled cortical impact in a rodent may induce memory deficits with prolonged cell death in the dentate gyrus (DG) of the hippocampus, few studies have reported long-term chronic hippocampal cell death following 'closed-head' TBI (cTBI), the predominant form of human TBI. Therefore, the aim of this study was to quantify terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)(+) apoptotic hippocampal cells as well as hippocampal cells with hallmark morphological features of degenerating cells in a chronic setting of cTBI in rats. TUNEL assays and Cresyl violet staining were performed using 6-month post-TBI fixed hippocampal sections. Evidence of prolonged hippocampal cell death was shown by the presence of a significantly increased number of TUNEL(+) cells in the cornu ammonis 1-3 (CA1-CA3) and DG of the hippocampus compared with intact controls. In addition, Cresyl violet staining indicated a significantly elevated number of cells with the degenerative morphological features in all hippocampal subregions (CA1-CA3, hilus, and DG). These results suggest that prolonged cell death may occur in multiple regions of the hippocampus following cTBI.

Insulin resistance in development and progression of nonalcoholic fatty liver disease.

Although insulin resistance (IR) is strongly associated with nonalcoholic fatty liver disease (NAFLD), the association of IR and NAFLD is not universal and correlation between IR and severity of NAFLD is still controversial. In this review, we summarize recent evidence that partially dissociates insulin resistance from NAFLD. It has also been reported that single-nucleotide polymorphisms in the diacylglycerol acyltransferase gene, rather than IR, account for the variability in liver fat content. Polymorphisms of the patatin-like phospholipase 3 gene have also been reported to be associated with NAFLD without metabolic syndrome, which suggests that genetic conditions that promote the development of fatty changes in the liver may occur independently of IR. Moreover, environmental factors such as nutrition and physical activity as well as small intestinal bacterial overgrowth have been linked to the pathogenesis of NAFLD, although some of the data are conflicting. Therefore, findings from both genetically engineered animal models and humans with genetic conditions, as well as recent studies that have explored the role of environmental factors, have confirmed the view that NAFLD is a polygenic disease process caused by both genetic and environmental factors. Therefore, IR is not the sole predictor of the pathogenesis of NAFLD.

Trigeminal neuroplasticity underlies allodynia in a preclinical model of mild closed head traumatic brain injury (cTBI).

Post-traumatic headache (PTH) following TBI is a common and often persisting pain disability. PTH is often associated with a multimodal central pain sensitization on the skin surface described as allodynia. However, the particular neurobiology underlying cTBI-induced pain disorders are not known. These studies were performed to assess trigeminal sensory sensitization and to determine if sensitization measured behaviorally correlated with detectable changes in portions of the trigeminal sensory system (TSS), particularly trigeminal nucleus, thalamus, and sensory cortex. Thermal stimulation is particularly well suited to evaluate sensitization and was used in these studies. Recent advances in the use of reward/conflict paradigms permit use of operant measures of behavior, versus reflex-driven response behaviors, for thermal sensitization studies. Thus, to quantitate facial thermal sensitization (allodynia) in the setting of acute TBI, the current study utilized an operant orofacial pain reward/conflict testing paradigm to assess facial thermal sensitivity in uninjured control animals compared with those two weeks after cTBI in a rodent model. Significant reductions in facial contact/lick behaviors were observed in the TBI animals using either cool or warm challenge temperatures compared with behaviors in the normal animals. These facial thermal sensitizations correlated with detectable changes in multiple levels of the TSS. The immunohistochemical (IHC) studies revealed significant alterations in the expression of the serotonin (5-HT), neurokinin 1 receptor (NK1R), norepinephrine (NE), and gamma-aminobutyric acid (GABA) in the caudal trigeminal nucleus, thalamic VPL/VPM nucleus, and sensory cortex of the orofacial pain pathways. There was a strong correlation between increased expression of certain IHC markers and increased behavioral markers for facial sensitization. The authors conclude that TBI-induced changes observed in the TSS are consistent with the expression of generalized facial allodynia following cTBI. To our knowledge, this is the first report of orofacial sensitization correlated with changes in selected neuromodulators/neurotransmitters in the TSS following experimental mild TBI.

Effect of telmisartan on histological activity and fibrosis of non-alcoholic steatohepatitis: A 1-year randomized control trial.

BACKGROUND/AIM: Telmisartan can attenuate two hit pathogenesis of non-alcoholic steatohepatitis (NASH). This study aimed to observe the effect of Telmisartan on non-alcoholic fatty liver disease (NAFLD) activity score (NAS) and fibrosis score in NASH patients. PATIENTS AND METHODS: A total of 50 NASH patients were randomized; 35 of group 1 were treated with Telmisartan 40/80 mg once daily with life style modification (TL) and 15 of group 2 underwent only life style modification (L) for 1 year. At the end, 20 of TL group and 10 of L group were analyzed. Those who showed NAS improvement ≥ 2 or NAS improvement ≥ 1 with fibrosis improvement ≥ 1 were considered as responders. RESULTS: Baseline alanine aminotransferase (ALT), aspartate aminotransferase (AST), insulin resistance index, components of metabolic syndrome, age, and sex were similar in both groups. At the end of study, NAS improvement in TL and L groups was 2.15 ± 1.66 and 1.10 ± 0.57 (P = 0.017) and fibrosis improvement was 0.65 ± 0.93 and -0.30 ± 0.48 (P = 0.001), respectively. NAS improved by ≥ 2 in 13 (65%) and 2 (20%) patients and fibrosis score improved by ≥ 1 in 8 (40%) patients and none of the patients in TL group and L group, respectively. Telmisartan and life style modification could improve steatosis, ballooning, lobular inflammation, and fibrosis. Life style modification could improve ballooning only, but fibrosis deteriorated. TL group showed improvement in NAS and fibrosis score [P value: 0.035; odds ratio (OR) =92.07, confidence interval (CI) =1.39-6106] to the level of response by regression analysis. Weight reduction and improvement of metabolic syndrome did not influence the response. There were similar minor adverse events in both groups. CONCLUSION: Telmisartan improved NAS and fibrosis score in NASH with insignificant adverse events.

Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A.

Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain.