NGS-based profiling reveals a critical contributing role of somatic D-loop mtDNA mutations in HBV-related hepatocarcinogenesis.

BACKGROUND: Somatic mutations of mitochondrial DNA (mtDNA) have been extensively identified mainly by traditional Sanger sequencing technology in various cancer types. However, low detection sensitivity of traditional methods greatly limits the comprehensive profiling of mtDNA somatic mutations in cancers, especially in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Moreover, the functional roles of mtDNA mutation in HBV-related hepatocarcinogenesis have not been systematically revealed. PATIENTS AND METHODS: Next-generation sequencing (NGS) platform was applied to profile the somatic mtDNA mutations of HCC and paired paratumor (non-HCC) tissues from a large cohort of 156 HBV-HCC patients. RESULTS: Our data revealed the common existence of mtDNA mutation in both inflammatory and cancer tissues with significantly different mutation pattern. The mutation density (mutation number/region length) of D-loop region was much higher than that of other regions in both HCC and non-HCC tissues. Unexpectedly, the average mutation number in D-loop region of HCC tissues was significantly less than that of non-HCC tissues. In contrast, the heteroplasmy level of D-loop region mutations was significantly increased in HCC tissues, implying that the D-loop mutations might be positively selected in HCC tissues. Furthermore, our results indicated that the patients with D-loop mutations had a significantly lower mtDNA copy number and were more likely to relapse. In vitro experiments demonstrated that proliferation, invasion and metastasis ability of HCC cells with D-loop region mutations were significantly higher than those without D-loop region mutations. CONCLUSION: These results emphasize the critical contributing role of somatic mtDNA D-loop mutations in HBV-related hepatocarcinogenesis.

Membrane current-based mechanisms for excitability transitions in neurons of the rat mesencephalic trigeminal nuclei.

Since Hodgkin's first description of three classes of excitability in crustacean nerve axons (1948), theoretical studies have used mathematical models to demonstrate that small changes in the parameters describing ionic currents could result in transitions between classes of membrane excitability. However, these transitions have rarely been investigated experimentally. Here, we show that states of excitability in rat mesencephalic V (Mes V) neurons can be classified into three groups, with manipulations of the 4-aminopyridine sensitive K(+) current (I(4-AP)) or persistent Na(+) current (I(NaP)) leading to the corresponding transitions. However, alterations in the hyperpolarization-activated cation current (I(h)), tetraethylammonium (TEA)-sensitive K(+) current, or Cd(2+)-sensitive Ca(2+) current were ineffective in causing these transitions. These results provide experimental evidence for the excitability transitions predicted by Hodgkin and characterize their ionic mechanisms in Mes V neurons.

High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer.

Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100 microg/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.

Synaptic transmission of chaotic spike trains between primary afferent fiber and spinal dorsal horn neuron in the rat.

Primary sensory neurons can generate irregular burst firings in which the existence of significant deterministic behaviors of chaotic dynamics has been proved with nonlinear time series analysis. But how well the deterministic characteristics and neural information of presynaptic chaotic spike trains were transmitted into postsynaptic spike trains is still an open question. Here we investigated the synaptic transmission of chaotic spike trains between primary Adelta afferent fiber and spinal dorsal horn neuron. Two kinds of basic stimulus unit, brief burst and single pulse, were employed by us to comprise chaotic stimulus trains. For time series analysis, we defined "events" as the longest sequences of spikes with all interspike intervals less than or equal to a certain threshold and extracted the interevent intervals (IEIs) from spike trains. Return map analysis of the IEI series showed that the main temporal structure of chaotic input trains could be detected in postsynaptic output trains, especially under brief-burst stimulation. Using correlation dimension and nonlinear prediction methods, we found that synaptic transmission could influence the nonlinear characteristics of chaotic trains, such as fractal dimension and short-term predictability, with greater influence made under single-pulse stimulation. By calculating the mutual information between input and output trains, we found the information carried by presynaptic spike trains could not be completely transmitted at primary afferent synapses, and that brief bursts could more reliably transmit the information carried by chaotic input trains across synapses. These results indicate that although unreliability exists during synaptic transmission, the main deterministic characteristics of chaotic burst trains can be transmitted across primary afferent synapses. Moreover, brief bursts that come from the periphery can more reliably transmit neural information between primary afferent fibers and spinal dorsal horn neurons.

A novel bursting mechanism of type a neurons in injured dorsal root ganglia.

Using intracellular recording in vivo, the bursting behaviors were investigated in the neurons of chronically compressed dorsal root ganglia of the adult rat. In most cases, the first spike of a burst emerged from amplitude-increasing damped subthreshold membrane potential oscillation (SMPO) and the discharge terminated by an amplitude-decreasing damped SMPO. The rhythms of these bursting behaviors are all irregular. Since some researchers found that the stochastic dynamics can also produce similar bursting pattern, the deterministic dynamics of interevent interval (IEI) series obtained from raw membrane potential recording was detected by extraction of the hierarchy of unstable periodic orbits (UPOs) in the windowed IEI series. The results showed a number of statistically significant UPOs of order-one, order-two, and order-three. These orbits form a complex but predictable lattice of regions in which the dynamics of the bursting occurrence is deterministic. Based on a complete classification scheme, the investigated bursting can be depicted by the elliptic bursting dynamics. The significance of the finding that a neuron in the injured dorsal root ganglion has such dynamics is also discussed.

Application of cell engineering technology to the tumour immunotherapeutic drug: a review.

The concept of cell engineering is frequently mentioned in recent years. It's a comprehensive technology which is very powerful and useful. By using it, scientists can produce many kinds of proteins and other biological moleculars in large scale that are in great demand for the human being. Among these products, most can be used for tumor immunotherapy and have been confirmed to be very effective in tumour control or subsidiary treatment. Of them, most common and useful drugs include three sorts; tumour vaccines, cell factors and monoclonal antibodies. The paper will discuss in detail related drugs application advances in tumour immunotherapy.

Subthreshold membrane potential oscillations of type A neurons in injured DRG.

An abundance of subthreshold membrane potential oscillations (SMPOs) at resting potential was observed in the neurons of chronically compressed dorsal root ganglia (DRG) using intracellular recording in vivo. Out of 386 neurons, 63 type A neurons displayed SMPOs. Three types of SMPOs were distinguished based on their characterizations of oscillation: (1) A regular pattern of SMPO emerged consistently with a mean frequency of 86 Hz and mean amplitudes of 3.3 mV. (2) A spindle-like pattern of SMPO was denominated by a spindle alteration of its amplitude. (3) An irregular pattern of SMPO had no rule on its change of amplitude and frequency. Compared with normal DRG neurons and injured DRG neurons but without SMPO, the injured DRG neurons with SMPO had the lowest spike rheobase, in accordance with the detection of spike accommodation. No significant differences among the three groups can be found in either membrane potential or input resistance. Further observation showed that the spontaneous discharge of hyperexcitable neurons usually occurred on the depolarizing phase of oscillations. In addition, the regular pattern of SMPO was based on the period and integer multiple patterns of spontaneous discharges. The spindle-like pattern of SMPO contributed to spontaneous bursting discharge. The irregular pattern of SMPO had a striking relation with irregular spontaneous discharge. The results show that neurons with SMPO in injured DRG have a higher excitability than those without SMPO, and that the SMPO underlie the patterns of spontaneous discharges, suggesting that SMPO is the basic electrophysiological change of hyperexcitable neurons.

Subthreshold membrane oscillations underlying integer multiples firing from injured sensory neurons.

Integer multiples firing (IMF), a special temporal pattern of firing, was recently observed in spontaneous discharge from injured dorsal root ganglion (DRG) neurons. To investigate the mechanism underlying IMF, the injured DRG neurons of rat were recorded intracellulary. Of 64 recorded A-neurons discharging spontaneously, eight fired spikes in the IMF pattern. Interspike interval (ISI) time series of IMF showed a structure of distinct bands on scatter map. Regular subthreshold membrane oscillations (SMOs) with relatively stable amplitude and frequency were observed on all eight IMF neurons. IMF could be induced from the neurons in periodic firing by local application of tetrodotoxin (TTX), a Na+ channel antagonist. During this process, the amplitude of SMOs varied markedly. Some SMOs were below action potential threshold so that they did not trigger spikes. Nor did some SMOs, though their amplitude were obviously beyond the threshold measured from nearby spikes. The results indicate that regular SMOs existent in injured DRG A-neurons underlie IMF, and the TTX-induced transformation of firing pattern from periodic to integer multiples may occur in two ways: decreasing the amplitude of SMOs and/or elevating action potential threshold.

Adrenergic sensitivity of neurons with non-periodic firing activity in rat injured dorsal root ganglion.

In this study, we compared the sensitivity of non-periodically and periodically active neurons in chronically compressed dorsal root ganglion in rats to norepinephrine and sympathetic stimulation. Forty-nine of 58 (84.5%) neurons with non-periodic activity showed responses to norepinephrine, whereas only five of 48 (10.4%) neurons with periodic activity displayed any response. The dose-response relationship of norepinephrine to the irregular burst pattern neurons shifted towards the left significantly compared to that of the periodic activity neurons. Responses to norepinephrine became apparent in eight neurons after their periodic firing activity was transformed into the non-periodic firing activity through the increase in Ca(2+). Changes in the time-response curves indicate a higher sensitivity of irregular burst pattern neurons to sympathetic stimulation than the periodic activity neurons. Finally, deterministic dynamics contained within the interburst interval series for non-periodic activity were identified. From these results, we suggest that the non-periodic activity neurons have a higher adrenergic sensitivity than those displaying periodic activity, and that this sensitivity may depend on the deterministic chaos within its firing dynamic system.

Relationship between calcium-dependent potassium channel and ectopic spontaneous discharges of injured dorsal root ganglion neurons in the rat.

The ectopic spontaneous discharges (ESD) of single myelinated dorsal root fiber originated from the injured dorsal root ganglion (DRG) neurons were recorded in vivo. When [Ca(2+)](o) perfusing the injured DRG had been enhanced or caffeine been used, the ESD was inhibited in dose-dependent manner, while using Ni(2+) or EGTA, the ESD facilitated. The increment of [K(+)](o) and the use of TEA could both facilitate the ESD in dose-dependent manner. Apamin, a special antagonist of calcium-dependent potassium channel (K(Ca)), had markedly increased the number of ESD. These results suggest that the generation of ESD has close relation to the activity of K(Ca) channel.

Ketanserin in the treatment of hypertension: a multicentre dose-finding study in China.

The effect of ketanserin at 5 mg, 10 mg and 20 mg twice daily for 3 months was studied in Chinese patients with hypertension. Both 10 mg and 20 mg doses twice daily effectively reduced systolic and diastolic pressures, while 5 mg twice daily was not effective. The 20 mg regimen was more effective than 10 mg in reducing diastolic, but not systolic pressures. Blood pressure reduction was progressive up to 1 month of treatment but not thereafter. Neither first-dose hypotension nor postural hypotension were seen. There were no effects on body weight or heart rate. A nominally significant 6 msec increase in QTc was seen with 10 mg, but not with 5 or 20 mg twice daily; this could be a chance finding. The side-effect burden was light, and decreased with time.