Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7 / 生物工程学报

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Comparison of the electromyographic activity of different zones of the abductor digiti minimi manus muscle in search of a functional compartmentalisation / Comparación de la actividad electromiográfica de diferentes zonas del músculo abductor digiti minimi manus en busca de una compartimentación funcional

SUMMARY: There is evidence demonstrating the presence of functional compartmentalisation (FC) in some skeletal muscles. This means that the motor units (MU), grouped in certain areas of the muscle, show different levels of activation in comparison to those located in other zones. This has only been described in large muscles whose morphology proves the existence of a FC. However, there is no background information about small muscles, such as the Abductor digiti minimi manus (ADM). The objective of this study was to compare the activation of the MU in different zones of the ADM to support the hypothesis of the existence of a FC in the ADM. By using a cross- sectional, analytical, observational study, the activity of the MUs in the ADM was assessed in 12 volunteers (age 21 ± 1.6 years old; weight 75.3 ± 8 kg; height 176.2 ± 7.3 cm; average ± standard deviation). The activity of MUs was evaluated using high-density surface electromyography (HD-sEMG) with an array of 64 electrodes arranged two-dimensionally. This allowed us to record the activity of the MUs in three zones of the ADM (Z1: dorsal zone; Z2: dorsal-palmar zone and Z3: palmar zone). Electromyographic recordings were obtained during voluntary isometric contractions of the ADM at 20, 40, 60 and 80 % of the maximum voluntary contraction (MVC). The comparison of the activation levels of MUs between the three zones was carried out using a mixed model analysis of covariance. The results showed a significant difference between the dorsal and palmar zones at 40 % of the MVC (p= 0.03), and between the dorsal and dorsal- palmar zone at 80 % of the MVC (p= 0.03). The results obtained in the evaluated sample support the hypothesis of the existence of FC in the ADM. However, further research is needed to determine with greater certainty the presence of this compartmentalisation in the ADM.

RESUMEN: Existe evidencia que demuestra la presencia de una compartimentalización funcional (CF) en algunos músculos esqueléticos. Aquello se traduce en que las unidades motoras (UM) agrupadas en ciertas zonas del músculo, presentan diferentes niveles de activación a las ubicadas en otras regiones. Esto solo ha sido descrito en músculos grandes, cuya morfología justifica la existencia de una CF. Sin embargo, no existen antecedentes de aquello en músculos pequeños, tales como el abductor digiti minimi manus (ADM). El objetivo de este estudio fue comparar la activación de las UM en distintas zonas del ADM, con la finalidad sostener la hipótesis de la existencia de una CF en el ADM. Mediante un estudio observacional analítico transversal se evaluó la actividad de las UM del ADM en 12 voluntarios (edad 21±1,6 años; peso 75,3±8 kg; altura 176,2 ± 7,3 cm; promedio ± desviación estándar). La actividad de las UM, se evaluó mediante electromiografía de superficie alta densidad usando una matriz de 64 electrodos dispuestos bidimensionalmente. Esta permitió registrar la actividad de las UM en tres zonas del ADM (Z1: zona dorsal; Z2: zona dorso-palmar y Z3: zona palmar). Los registros electromiográficos fueron obtenidos durante contracciones isométricas voluntarias del ADM al 20, 40, 60 y 80 % de la contracción voluntaria máxima (CVM). La comparación de los niveles de activación de las UM entre las tres zonas fue realizada mediante un análisis de modelos mixtos de covarianza. Los resultados indicaron que existió diferencia significativa entre la zona dorsal y palmar al 40 % de la CVM (p=0,03), y entre la zona dorsal y dorso-palmar al 80 % de la CVM (p=0,03). Los resultados obtenidos en la muestra evaluada sostienen la hipótesis de la existencia de una CF en el ADM. Sin embargo, son necesarias más investigaciones para establecer con mayor certeza la presencia de esta compartimentalización en el ADM.

Neuromuscular compartmentalization of the vastus medialis muscle: comparison of the activity of the vastus medialis obliquus and the vastus medialis longus by high density electromyography / Compartimentación neuromuscular del músculo vasto medial: comparación de la actividad del vasto medial oblicuo y del vasto medial largo mediante electromiografía de alta densidad

SUMMARY: Anatomical studies describe the vastus medialis (VM) as being subdivided into two morphologically distinct components, the vastus medialis obliquus (VMO) and the vastus medialis longus (VML). However, there are discrepancies regarding the functional differentiation of these components. The aim of this study was to compare the levels of activation of the VMO and the VML by high density surface electromyography. Twelve healthy young women (age: 21.4 ± 2.0 years; weight: 58.1 ± 7.5 kg; height: 1.6 ± 0.1 m), performed an open kinetic chain knee exercise during which the EMG activity of the VMO and the VML was recorded with two- dimensional matrices of 32 surface electrodes. The exercises were performed with three levels of resistance (5, 10 and 15 % of the body weight (BW)), considering three phases: concentric, isometric and excentric. In the isometric phase the VMO had greater activation than the VML with the three levels of resistance (p<0.05). In the excentric phase, the VMO also showed greater activation than the VML with the 10 and 15 % BW resistance levels, while in the concentric phase, the VMO showed greater activity than the VML with only the 15 % BW resistance. The results indicated significant differences in the activation level of the two components of the VM. This bears importance in the development of exercises intended to achieve a greater or more selective activation of the VMO. In the sample subjected to evaluation, the EMG recordings describe a greater activation of the VMO in comparison to the VML, which is more important in the isometric and excentric phases of the flexion/extension of the knee in an open kinetic chain. These findings suggest a functional compartmentalization of the VM.

RESUMEN: Los estudios anatómicos describen que el músculo vasto medial (VM) se subdivide en dos componentes morfológicamente distintos, el vasto medial obliquus (VMO) y el vasto medial largo (VML). Sin embargo, existen discrepancias con respecto a la diferenciación funcional de estos componentes. El objetivo de este estudio fue comparar los niveles de activación del VMO y el VML mediante electromiografía de superficie de alta densidad. Doce mujeres jóvenes sanas (edad: 21,4 ± 2,0 años; peso: 58,1 ± 7,5 kg; altura: 1,6 ± 0,1 m), realizaron un ejercicio de rodilla de cadena cinética abierta durante el cual se registró la actividad EMG de la VMO y la VML con dos matrices dimensionales de 32 electrodos de superficie. Los ejercicios se realizaron con tres niveles de resistencia (5, 10 y 15% del peso corporal (PC)), considerando tres fases: concéntrica, isométrica y excéntrica. En la fase isométrica el VMO tuvo mayor activación que el VML con los tres niveles de resistencia (p <0,05). En la fase excéntrica, el VMO también mostró mayor activación que el VML con los niveles de resistencia de 10 y 15% BW, mientras que en la fase concéntrica, el VMO mostró mayor actividad que el VML con solo el 15 % de resistencia al BW. Los resultados indicaron diferencias significativas en el nivel de activación de los dos componentes de la VM. Esto tiene importancia en el desarrollo de ejercicios destinados a lograr una activación mayor o más selectiva del VMO. En la muestra sometida a evaluación, los registros EMG describen una mayor activación del VMO en comparación con el VML, que es más importante en las fases isométrica y excéntrica de la flexión / extensión de la rodilla en cadena cinética abierta. Estos hallazgos sugieren una compartimentación funcional de la VM.

Exo70 intracellular redistribution after repeated mild traumatic brain injury

BACKGROUND: Exo70 is a subunit of the greater exocyst complex, a collection of proteins that oversees cellular membrane addition and polarized exocytosis by acting as a tethering intermediate between the plasma membrane and newly synthesized secretory vesicles. Although Exo70 function has been implicated in several developmental events including cytokinesis and the establishment of cell polarity, its role in neuropathologies is poorly understood. On the other hand, traumatic brain injury is the result of mechanical external force including contusion, fast acceleration, and expansive waves that produce temporal or permanent cognitive damage and triggers physical and psychosocial alterations including headache, memory problems, attention deficits, difficulty thinking, mood swings, and frustration. Traumatic brain injury is a critical health problem on a global scale, constituting a major cause of deaths and disability among young adults. Trauma-related cellular damage includes redistribution of N-methyl-D-aspartate receptors outside of the synaptic compartment triggering detrimental effects to neurons. The exocyst has been related to glutamate receptor constitutive trafficking/delivery towards synapse as well. This work examines whether the exocyst complex subunit Exo70 participates in traumatic brain injury and if it is redistributed among subcellular compartments RESULTS: Our analysis shows that Exo70 expression is not altered upon injury induction. By using subcellular fractionation, we determined that Exo70 is redistributed from microsomes fraction into the synaptic compartment after brain trauma. In the synaptic compartment, we also show that the exocyst complex assembly and its interaction with GluN2B are increased. Finally, we show that the Exo70 pool that is redistributed comes from the plasma membrane. CONCLUSIONS: The present findings position Exo70 in the group of proteins that could modulate GluN2B synaptic availability in acute neuropathology like a traumatic brain injury. By acting as a nucleator factor, Exo70 is capable of redirecting the ensembled complex into the synapse. We suggest that this redistribution is part of a compensatory mechanism by which Exo70 is able to maintain GluN2B partially on synapses. Hence, reducing the detrimental effects associated with TBI pathophysiology.

Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms andmicro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

Rapid generation of double-layer emulsion droplets based on microfluidic chip / 生物工程学报

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.

Caracterización morfológica del músculo bíceps femoral del perro / Morphological characterization of biceps femoris muscle in the dog

A partir de estudios previos realizados sobre la anatomía del musculo bíceps femoral en el perro y con el objetivo de avanzar en la caracterización inmunohistoquímica y nerviosa de este musculo, estudiamos la existencia de subvolúmenes musculares. Utilizamos la disección roma en piezas musculares aisladas fijadas y tratadas con ácido nítrico al 25%. Las divisiones de las ramas del nervio isquiático involucradas fueron utilizadas como guía en la disección. Se determinaron tres subvolúmenes, uno involucrando a la cabeza caudal y dos en el vientre correspondiente a la cabeza craneal que fueron denominados caudal, proximal y distal respectivamente. Realizamos mediciones del largo de las fibras musculares, dirección, dirección y densidad macroscópica de las mismas para caracterizar los compartimientos. Estos subvolúmenes deberán tenerse en cuanta al realizar estudios electromiográficos del musculo bíceps femoral y al evaluar las alteraciones en la locomoción producto de las alteraciones nerviosas y musculares en el nervio pelviano del canino.

From previous studies on the anatomy of the femoral biceps muscle in the dog and with the aim of advancing in immunohistochemical and nervous characterization of this muscle, we studied the existence of subvolumes in muscle structure. We used blunt dissection in isolated muscle pieces fixed and treated with 25% nitric acid. The divisions of the sciatic nerve branches involved were used to guide the dissection. Three subvolumes were identified; one, involving the caudal head, and two in the cranial head belly; they were called caudal, proximal and distal respectively. We measured muscle fiber length, orientation, and macroscopic density to characterize these compartments. These subvolumes must be considered when muscle electromyography research is carried out, and for locomotion changes evaluation due to lesions in nerve and muscle structure of the canine pelvic limb.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

Spatial and temporal variation of Dona Francisca reservoir (Jacuí river, Rio Grande do Sul State), a subtropical reservoir / Variação temporal e espacial do reservatório Dona Francisca (rio Jacuí, Estado do Rio Grande do Sul), um reservatório subtropical

The present study examined the behavior of a reservoir located on subtropical latitude (Dona Francisca reservoir) . The limnological variables measured were dissolved oxygen, temperature, pH and electric conductivity. Measurements were taken quarterly at different sampling sites along the reservoir. The results indicated the formation of longitudinal zones regarding circulation patterns and stratification, beyond the influence from the water masses of the main tributary. The vertical profiles were obtained. The behavior of the reservoir is similar to others previously studied in the country, but with shorter time-lag stratification and a narrower range, possibly due to the shorter residence time of the water, and to the latitude, where summer is shorter.

O estudo buscou analisar o comportamento das variáveis limnológicas, dentre elas oxigênio dissolvido, temperatura, pH e condutividade elétrica, em um reservatório de latitude subtropical (reservatório Dona Francisca). Medidas trimestrais foram feitas em diferentes locais do reservatório, onde foram obtidos os perfis de profundidade. Os resultados indicaram a formação de zonas longitudinais a partir dos padrões de circulação e estratificação, além da influência exercida pelas massas de água do afluente principal. Conclui- se que o comportamento do reservatório se assemelha a outros no país, mas com intervalo de tempo de estratificação menos prolongado e amplitudes menos acentuadas, em parte, possivelmente, pelo pequeno tempo de residência da água e sua posição latitudinal, quando o verão é mais curto.

STUDY ON COMPARTMENTALIZATION, DIS-TRIBUTION AND CROSS-SECTIONAL AREAS OF FIBER TYPES IN HUMAN DELTOID MUSCLE / 解剖学报

The deltoid muscles on both sides of 8 male cadavers were studied within 24 hours after death. According to its fiber architecture, origins and insertions, and nerve distribution, the deltoid muscle was divided into three compartments: an anterior, a middle and a posterior. The muscle samples were taken respectively from the superficial and the deep portion of each compartment of the deltoid muscle. Frozen transverse sections were stained fer myosin ATPase. Muscle fibers were identified as type Ⅰ and type Ⅱ. The proportion of fiber types in each portion was calculated on microscopic photographs and mean cross-sectional areas of both fiber types were measured by imaging analytical system. The results showed that the proportion of type Ⅰ fiber in the superficial and deep portion of the middle compartment is significantly higher than that of the corresponding portion of the anterior and posterior compartments, and the proportion in the deep portion of each compartment is higher than that of the superficial one, however no difference was found between the left and right sides. The fiber size among all portions is not obviously different, but the diameter of type Ⅱ fiber in the deep portion of the middle compartment is significantly smaller than that of other portions. Except the deep portion of the middle compartment, the diameter of both fiber types of the right muscle is larger than those of the left one, although statistical analysis showed that the difference is not significant. The authors suggest that the difference of fiber type distribution within the deltoid muscle is closely related with the functional differentiation of the muscle and the characteristic higher typeⅠ fiber proportion and smaller type Ⅱ fiber diamiter in the deep portion of the middle compartment probably indicate that the main function of this portion is to keep the shoulder joint stability.