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醫學與健康科學

Development of a neurointerface glove with tactile feedback

Research Question or Engineering Problem A stroke continues to be the most important medical and social problem of the modern world. Stroke is a type of acute cerebrovascular accident (ACVA) and is characterised by a sudden (within minutes, less often - hours) appearance of focal neurological symptoms (motor, speech, sensory, coordinating, visual and other disorders) and / or general brain disorders (depression of consciousness, headache, vomiting, etc.) that persist for more than 24 hours or lead to death of the patient in a short period of time due to a cause of cerebrovascular origin. There are two clinical and pathogenetic forms of stroke: ishemic stroke (cerebral infarction) is caused by acute focal cerebral ischemia, leading to infarction (zone of ischemic necrosis) of the brain; hemorrhagic stroke (non-traumatic intracerebral hemorrhage) is caused by rupture of an intracerebral vessel and blood penetration into the brain parenchyma or rupture of an arterial aneurysm with subarachnoid hemorrhage (SAH). ACVA also includes transient disorders of cerebral circulation, characterised by the sudden occurrence of focal neurological symptoms that develop in a patient with cardiovascular disease (arterial hypertension, atherosclerosis, atrial fibrillation, vasculitis, etc.), last for several minutes, less often hours, but no more than 24 hours, and ends with a full restoration of the impaired brain functions. Transient disorders of cerebral circulation include: transient ischemic attack (TIA), which develops as a result of short-term local cerebral ischemia and is characterised by sudden transient neurological disorders with focal symptoms; hypertensive cerebral emergency, which is a condition associated with an acute (usually significant) rise in blood pressure (BP) and accompanied by the appearance of general cerebral (less often focal) neurological symptoms secondary to hypertension. The most severe form of hypertensive crisis is acute hypertensive encephalopathy, the basis of pathogenesis of which is cerebral edema. Cerebral infarction generally is the result of the interaction of many etiopathogenetic factors, which can be subdivided into local and systemic ones. Local factors include: morphological changes in the brachiocephalic or intracerebral arteries (pathological tortuosity, etc.), atherosclerotic lesions of the vessels of the aortic arch and cerebral arteries, cardiac lesions as a source of thromboembolic cerebral infarctions, fibromuscular dysplasias of the walls of the brachiocephalic and cerebral arteries, brachiocephalic artery dissection, vasculitis (arteritis), changes in the cervical spine with the formation of extravasal compression of the vertebral arteries, anomalies in the structure of the vessels of the neck and brain (hypoplasia of the vertebral artery, trifurcation of the internal carotid artery), etc. Systemic factors include: disorders of central and cerebral hemodynamics (a sharp change in BP or a decrease in cardiac output, etc.), hereditary and acquired coagulopathies, polycythemia, certain forms of leukemia, hypovolemia, psychoemotional stress / distress, etc., hypercoagulative / hyperaggregatory side effects of a number of medications (oral contraceptives, etc.). In the 俄羅斯n Federation, more than 500 thousand people have a stroke every year. About 25,000 new cases of stroke occur in St. Petersburg every year. The incidence of stroke in the 俄羅斯n Federation is 3.48 ± 0.21 cases per 1000 people. The incidence of various types of ACVA varies widely, in particular, cerebral infarctions account for 65–75%, hemorrhages (including subarachnoid hemorrhages) – 15–20%, transient cerebral circulation disorders account for 10–15%. The frequency of cerebral strokes in the population over 50–55 years old increases by 1.8–2 times in each subsequent decade of life. Medical and socio-economic consequences of ACVA are very significant, in particular, death in the acute period of stroke occurs in 34.6% cases, during the first year after the end of the acute period in 13.4% cases; severe disability with the need for constant care is present in 20.0% of stroke patients; 56.0% have limited working capacity and only 8.0% return to their previous work activity. Disability due to stroke (the national average is 56–81%) in our country ranks first among all causes of primary disability, amounting to 3.2 per 10 thousand people. Stroke mortality among working-age population has increased in the 俄羅斯n Federation by more than 30% over the past 10 years. The annual death rate from stroke in our country is 175 per 100 thousand people. Stroke annually becomes the main cause of disability: 85% of victims experience a decrease in strength or a complete lack of ability to control the muscles of half of the body and only half of them recover limb functions partially or completely; the rest of those who have suffered a stroke remain paralysed and require care, since they are not able to completely independent existence. In this regard, recently, in the process of rehabilitation, the technology of brain-computer interfaces (BCI) has begun to be actively used. on the basis of this technology exercise machines are created. These exercise machines are controlled directly by the patient himself. This feature of the technology increases the effect of the procedure by providing a direct connection between the patient's desire and effort with the work of the simulator. The greatest development of this technology is observed in the field of medicine, where BCIs are used as a means of communication or as one of the tools of neurorehabilitation. In this regard, it seems very promising to develop the most optimal brain-computer interfaces. The goal of our project was to create an automated training complex in the form of a neuro-controlled glove with tactile feedback, designed to simplify access to rehabilitation means.

Lighting Up The Brain

Alzheimer’s disease (AD) is a neurodegenerative disease in which current diagnostic tools are invasive and lack the ability to diagnose early-onset dementia. Current antibody-based diagnostic tests for neurodegenerative diseases require invasive measures such as a lumbar puncture, and lack specificity to biomarkers that are found in both healthy individuals and patients with AD. In this project, a design for a carbon dot(CD)-bound bispecific antibody is developed for the minimally- invasive diagnosis of AD. The molecular probe can be easily synthesized with a specificity to amyloid- beta (Aβ) oligomers as it distribution and abundance in the brain suggest they are better predictors of disease progression and are present in the early-onset of the dementia. The bispecific antibody conjugated to the CD displays a low affinity to transferrin receptors (TfRs) which allows the probe to cross the blood-brain barrier via receptor mediated transcytosis leading to a minimally invasive diagnosis. A synthesis technique was developed to conjugate the bispecific antibody to the CD. As a proof of concept, this technique was used to couple bovine serum albumin (BSA) to CDs. The structural and optical properties of the CDs were observed. By synthesizing a novel carbon dot conjugated specific antibody that emits light at a specific wavelength in the near-infra red region, the molecular probe displays optical properties suitable for the minimally-invasive diagnosis using fNIR- spectroscopy.

新式高能磁場對於骨髓幹細胞軟骨分化的影響

近年來,再生醫療利用幹細胞幫助組織器官新生的方法已有長足進步,但仍然面臨許多的瓶頸。例如: 幹細胞注射進入人體以後,無法繼續分化生長成所需組織。最近,高能磁場的使用也是醫學界蓬勃發展的議題,我們試圖利用高能磁場誘導幹細胞的分化,希望可以增加幹細胞分化的效能,這是一創新的嘗試。我們比較新型高能磁場(RMS)與對照組於骨髓幹細胞(BMSCs)的分化效果,發現RMS 中叢集式刺激(iTBS)的SOX9和COL10的基因表現與免疫組織化學染色明顯優於15Hz RMS 與對照組。最佳的刺激設計為強度50%的iTBS連續施打五天。我們亦發現每回合900 pulses有優於每回合600 pulses的趨勢。此實驗證實iTBS能夠增加BMSCs在體外的軟骨分化表現,反映在基因表現層次到膠原蛋白合成的層次。未來,此方法將進一步使用於動物實驗,期望擴展到臨床上用於退化性關節炎的治療,對再生醫療發展有所幫助。

Cellular Effects of DNA Demethylation Enzyme TET2 Knockdown

近年來許多研究指出TET家族應是抑癌基因,然其許多抑癌機制卻尚未明瞭。根據本實驗室未發表的實驗結果,TET2表現量在大多乳癌檢體內較低,且TET2表現量低之病患存活率較差。本研究以Western Blot發現H3k4甲基化量在TET2 knockdown後減少。實驗室microarray結果更發現重要乳癌抑癌基因DKK1在TET2 knockdown後表現量下降。本研究以real-time PCR進一步證實DKK1表現量下降;Western Blot與ELISA結果也顯示DKK1蛋白質在細胞內及培養液中減少。細胞實驗更發現TET2 knockdown及DKK1 knockdown皆導致細胞有更顯著的移行(migration)與增生(proliferation)特性,因此推測DKK1可能為受TET2調控的下游基因。已知DKK1轉譯出之蛋白質能拮抗抑制Wnt/β-catenin傳遞路徑,因此本研究正在探討TET2是否也調控此Wnt路徑。如證實此假設,將可提出TET2操控DKK1,DKK1調控Wnt路徑,終而影響EMT、癌症轉移的路徑。

MafF對LRH-1調控代謝機制之探討

已知 liver receptor homolog-1(LRH-1)掌控著多種生理功能,在肝臟代謝中扮演著相當重要的角色,我們利用酵母菌雙雜交技術(Yeast two-hybrid)找出數種可能跟LRH-1產生交互作用的蛋白質,並從其中選擇v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F (MafF)作為主要研究對象,探討其與LRH-1之間的作用對代謝造成的影響。本研究中,我們利用免疫沉澱法證實MafF可與LRH-1形成複合體,而MafF並不影響LRH-1蛋白的表現量。隨後以啟動子活性檢測MafF-LRH-1複合體在細胞內之功能,發現MafF能促進由LRH-1所調控的small heterodimer partner (SHP)、glucokinase IV (Gck)及side chain cleavage enzyme (SCC)等代謝相關啟動子之活性。此外我們以GST-pull down發現MafF與LRH-1結合區域位於LRH-1的DNA 鍵結位(DBD)。LRH-1則是僅與具有完整basic region (BR)以及leucine zipper (LZ) 兩個domain的MafF產生交互作用,單獨的BR以及LZ無法與LRH-1結合。另外,在肝臟細胞株HepG2中過度表現Myc-MafF,Western blot結果發現GCK有減少的趨勢,SHP則是有增加的現象,而LRH-1亦呈現增多的趨勢。Real-time PCR結果,比對Myc以及Myc-MafF,發現MafF存在時會使LRH-1以及GCK mRNA含量較控制組Myc有減少的趨勢,對SHP mRNA 則是沒有顯著影響。此外,我們在肝臟細胞株HepG2中加入胰島素,發現二者表現量皆有明顯的增加,說明胰島素刺激之下,MafF以及LRH-1蛋白質增加能夠增強肝臟能量代謝。

探討雙黃酮類CRYT對抑制發炎反應與免疫細胞移動的影響

發炎反應與癌症具有密切的關係,而如何抗發炎是現今重要的研究主題。本研究主要探討雙黃酮類藥物CRYT對LPS誘發之發炎反應與免疫細胞遷移的影響。本研究首先以細胞試驗分析細胞激素與趨化因子的表現量觀察CRYT對發炎反應的影響。接著,利用反轉錄聚合酶連鎖反應 (RT-PCR) 和共軛焦顯微鏡觀察黏附分子與調控細胞骨架分子的表現量,以了解CRYT對免疫細胞遷移的影響。再來我們以西方墨點法分析免疫反應調控路徑之蛋白質表現量來研究CRYT的作用途徑。同時,透過程式模擬CRYT的標的蛋白,並用細胞轉染與雙冷光技術驗證模擬結果。本研究發現,CRYT會抑制TNF-、IL-1及IL-6三種促發炎因子與CXCL1、CCL3及CCL4三種趨化因子的表現量。另外,CRYT會抑制黏附分子LFA-1與VLA-4與調控細胞骨架分子Cdc42、PAK1、WASP、Rac1及LIMK1之mRNA表現量,以阻礙F-actin進行聚合作用。本研究也發現,CRYT能藉由抑制NF-B的表現量,進而影響巨噬細胞的發炎作用與細胞移動。在作用機制的方面,CRYT會結合在雌激素受體的配體結合位,並抑制雌激素受體的轉錄作用,而抑制發炎作用與細胞轉移。結論得知,CRYT不但可以抑制發炎反應,還能有效抑制免疫細胞的遷移。

THE DESIGN OF MICROFLUIDIC PUMP (MFP) FOR MEDICAL FIELD

The ability of microfluidic (MF) device technologies to provide a lot of information with a small amount of sample, the opportunities it offers increases their use in the medical field in the bedside monitoring in drug delivery systems. Three-dimensional (3D) printer technologies provide advantages such as cost-effectiveness in the production of MF devices and quick and easy production in intricate designs. In our project, it is aimed to design microfluidic pumps (MFP) to be used in the medical field and conduct its production with 3D printer technologies. The developed MFP is intended to be at low cost, bio-compatible, adaptable, and portable to the drug, suitable flow properties as a pharmaceutical pump. First of all, MFP air channel, flow channel, etc. parts were designed and printed with the help of a 3D printer and on AutoCAD, one of the professional drawing programs. The poly(dimethylsiloxane) (PDMS) membrane that will enable MFP activation is produced in different thicknesses and glued to the air channel of MFP. The resistance to the applied pressure is observed, and the appropriate membrane thickness is determined as ~ 235µm. Liquid PDMS was applied to the inner surfaces of MFP's air and flow channel, PDMS membrane was placed between them, and the parts were assembled in the oven at 60ºC. MFP has been connected to the pneumatic valve system, where operation codes have been prepared with Arduino Uno, and flow properties have been examined. The flow rate of MFP is ~ 50 µL/min at a maximum of 15 Hz, and the backpressure is ~ 0.085 Pa under a maximum pressure of 3 bar. Also, values such as size, membrane thickness, and applied pressure for the possible models of MFP were supported by theoretical calculations. As a result, MFP, which is biocompatible, drug adaptable, portable, wearable technology application potential, and has suitable flow characteristics as a pharmaceutical pump, has been developed. MFP introduced a microfluidic pump system that can make life easier for the patient and contribute to the national economy through domestic production and can be used as a drug pump in the treatment of diseases such as diabetes and cancer.

HOST TARGET PROTEINS OF SPIKE PROTEIN OF SARS-COV-2

Coronavirus Disease 2019 (COVID-19) is a newly emerged infectious disease caused by the new severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-2). In less than one year, the virus has spread around the entire world, killing millions of people and disrupting travel and business worldwide. During infection, the virus uses its Spike protein to dock onto the Ace2 protein on the surface of its human host cell. Spike is 1273 amino acids long and only a short fragment of Spike (319-541) is sufficient to bind Ace2. We hypothesized that the remaining protein sequences of Spike might have functions for viral replication beyond the binding of Ace2. We have performed Split-Ubiquitin protein-protein interaction screens to isolate human proteins by their ability to bind to Spike, and we have identified Annexin2A2 and Cytochrome b as novel human protein interaction partners of Spike. Annexin2A2 is involved in both endocytosis and exocytosis, and the protein interaction with Spike might help the virus to enter and exit its host cell. The presence of the mitochondrial Cytochrome b protein inside the cytosol promotes apoptosis, and the protein interaction with Spike could speed up sapoptosis of the infected human cell. The Nub cDNA libraries that we have generated also allowed us to screen for synthetic peptides that interact with Spike. We have isolated two synthetic peptides, FL1a and FL7a, derived from the non-coding parts of human mRNAs by their ability to interact with Spike. We found that both FL1a and FL7a interact with the C-terminal half of the Spike protein. We also found that FL7a is able to block the Spike-Spike self-interaction at the C-terminal half of the Spike protein and we think that this could block the reassembly of the Spike protein in the host cell during viral reassembly. We hope that those synthetic peptides could be used as drugs due to their ability to block protein-protein interactions of Spike with human host proteins that are essential for viral replication.

探討吸菸調控絲胺酸合成路徑影響肺癌生長及抗藥性

肺癌為全球死亡率最高的癌別之一。抽菸,肺癌的主要危險因子,臨床上造成抗癌藥效不佳並導致病患的低存活率與不良預後。然而,抽菸影響肺癌的機制仍不清楚。代謝重整最近被視為是癌症的新興特點。絲胺酸合成路徑為葡萄糖代謝的分支之一,參與生物合成材料之製造,並和癌症的惡化有密切的關連性,但缺乏詳細的相關研究。本研究探討抽菸是否透過影響絲胺酸合成路徑來導致肺癌生長,並測試絲胺酸合成路徑抑制劑是否能增強化療藥物吸菸相關肺癌細胞的治療效果。我們的研究發現,抽菸和絲胺酸合成路徑在肺癌中有正相關性且與臨床上的低存活率有關,並證實抽菸調控絲胺酸合成路徑而促進肺癌生長與化療抗性,此現象可因合併給予絲胺酸合成路徑抑制劑而獲得緩解。此研究成果顯示抑制絲胺酸合成路徑可能成為治療吸菸相關肺癌的新策略。

Preparation of a Specific Detector for Aspergillus Niger in Swimming Pools

Swimming pools are one of the transmission routes of superficial and cutaneous fungal infections. Maintenance of environmental hygiene in different parts of swimming pools is of great importance, especially the hygiene of water (1). The conventional fungal detection methods include direct smear preparation, culture, and pathological examinations. However, these methods are not fast enough or do not have sufficient sensitivity (2). Therefore, the present research introduces a novel method for detecting Aspergillus niger in pool water through creating optimal conditions for this fungus, which leads to the citric acid production by the fungus and pH changes of the related culture medium. Four experiments in 10 steps were performed to find the optimal conditions for fungal growth. According to our results, adding each of the variables sucrose, soy, and ferrous sulfate can lead to favorable results. Moreover, the shaker speed increase and fungal aeration are important. Also, we showed that soybean led to the best results compared to other variables. Considering the obtained results, including the shortened detection duration and cost-effectiveness, this method can be presented to the swimming pool owners and pathobiology laboratories as the method of choice for Aspergillus niger detection.