EdU体外标记毛囊外根鞘干细胞的实验研究

摘要 目的：探寻一种简单有效的追踪定位毛囊干细胞的分裂增殖标记的方法,为后期毛囊干细胞增殖分化迁移机制的研究打下基础。方法：体外无菌条件下获取昆明乳鼠触须毛囊外根鞘,以5 % FCS+MEM作为基础培养液,添加4种不同浓度EdU（5-Ethynyl-2’-deoxyuridine）细胞标记物,进行毛囊外根鞘再生培养,冰冻切片,H.E染色,使用KeyFluor488对EdU所标记的再生细胞进行检测。结果：H.E染色和EdU标记染色结果发现EdU细胞标记物浓度越高,毛囊外根鞘再生形态越差,同时当EdU浓度为20 μ mol/L时,毛囊外根鞘增殖细胞标记效果是最好的。结论：EdU细胞标记物对毛囊外根鞘中的干细胞增殖活性具有一定的毒副作用,可能会影响细胞的正常生理代谢功能,浓度为20 μ mol/L EdU可以有效的标记毛囊干细胞增殖,并对毛囊再生结构影响较小。     

新化合物大麻二酚衍生物CBD-S的合成及体外活性研究

摘要 目的：大麻二酚（CBD）具有抗肿瘤和抗炎活性,是一种非成瘾性的活性成分,近年来受到医药界的广泛关注,但其水溶性较差,限制了其临床应用。本文拟合成一种新型的水溶性化合物,并测试其抗肿瘤与抗炎活性。方法：本文主要基于CBD进行结构改造,化学合成了新化合物大麻二酚2,6-苯氧乙酸二钠盐（CBD-S）,通过核磁共振氢谱、碳谱和高分辨质谱对其化学结构进行了表征,通过高效液相色谱（HPLC）方法测试了其溶解性。同时在HepG2(人肝癌细胞)和J774A.1(小鼠单核巨噬肿瘤细胞系)细胞上分别通过噻唑蓝（MTT）和Western blotting的方法测试了其体外抗细胞增殖及抗炎活性。结果：我们成功获得了CBD-S新化合物,氢谱、碳谱和高分辨质谱鉴定结果证实新合成的CBD-S结构正确,HPLC方法测试结果显示该化合物具有较好的水溶性。在细胞增殖实验中,我们发现该化合物在细胞培养液中终浓度为5-10 μg/mL时,以剂量依赖性方式显著抑制人肝癌HepG2细胞的增殖。在细胞抗炎活性研究中,为了获得在J774A.1细胞上的安全无毒浓度来开展后续的活性测试,我们首先检测了该化合物在J774A.1细胞上的毒性,发现在该细胞系温孵CBD-S在细胞培养液中终浓度为10 μg/mL以下时,是没有细胞毒的。接下来,在J77A.1细胞,我们发现CBD-S在细胞培养液中终浓度为0.01-1.00 μg/mL时,显著抑制了脂多糖（LPS）诱导的环氧合酶-2（COX-2）蛋白的生成。结论：新合成的化合物CBD-S具有较好的水溶性,在体外具有一定的抗肿瘤与抗炎活性,提示其具有较好的应用前景,值得进一步的深入或体内验证研究。     

大叶山楝根化学成分与细胞毒活性的研究

为了解大叶山楝(Aphanamixis grandifolia Bl.)根中的抗肿瘤活性成分,利用各种色谱技术从其95% 乙醇提取物中分离得到9 个化合物,经波谱分析分别鉴定为:7-hydroxycadalene (1)、dregeana-1 (2)、4-oxopinoresinol (3)、4-ketopinoresinol (4)、6-deoxyjacareubin (5)、schleicheol 1 (6)、豆甾醇 (7)、β-谷甾醇 (8)和胡萝卜苷 (9).其中化合物1~6 为首次从山楝属植物中分离得到,并首次报道了化合物1的碳谱数据.生物活性测试结果表明,化合物1和5对慢性髓原白血病细胞K562 有生长抑制活性,化合物1、5 和6 对人胃癌细胞SGC-7901 有生长抑制活性.     

31P-NMR研究生物活组织

NMR在生物学中的应用是从70年代发展起来的。 ³¹P-NMR法因其共振谱线简单明了,灵敏度高,能无损伤地监控生物活组织内的与能量代谢密切相关的含磷化合物浓度的动态变化,而成为在生理条件下测定组织内部动力学参量的重要方法。本文通过介绍离体灌流标本和在体组织器官的 ³¹P-NMR的具体应用实例,集中反映了在这一领域研究的概况和新近的研究成果。     

胎盘脱细胞基质作为组织修复材料的初步研究

目的 制备胎盘脱细胞基质并评价其生物相容性，探讨其作为组织修复材料的可行性。方法 利用分娩废弃物胎盘组织，进行病毒灭活、脱细胞处理、冷冻干燥得到胎盘脱细胞海绵状基质材料，HE染色观察脱细胞效果及扫描电镜观察材料微观结构。同时，选取健康雄性SD大鼠39只，体质量120～150 g，随机分为实验1组、实验2组及对照组。对构建的基质材料进行大鼠皮下植入实验，实验1组植入基质材料，实验2组植入基质材料及脐带间充质干细胞，对照组为假手术组。于手术后第3、5、7天进行动物血常规检测，分析淋巴细胞、粒细胞等炎性细胞数量；于手术后第1、2、4、8、9周取材料植入处及周围组织样本进行HE染色分析。结果 构建的胎盘脱细胞基质肉眼观呈乳白色海绵状，HE染色未见细胞残留，电镜观察材料内部空隙比较明显，材料交联度较好，总孔隙率为（77.54±2.53）%。皮下植入之后，切口处愈合良好，血常规结果未见明显的炎性细胞增多；术后7 d植入材料切片HE染色即可见血管形成，且脐带间充质干细胞的加入能够加快材料与机体的融合，促进细胞的深入生长及血管化。结论 胎盘脱细胞海绵基质材料具有良好的生物相容性，可作为组织工程材料的理想来源。     

BIS监测应用于七氟醚吸入全身麻醉对老年腹腔镜胆囊切除术患者苏醒质量、应激反应和认知功能的影响

摘要 目的：观察脑电双频指数（BIS）监测应用于七氟醚吸入全身麻醉对老年腹腔镜胆囊切除术（LC）患者苏醒质量、应激反应和认知功能的影响。方法：选择2018年4月~2021年6月期间我院收治需全麻下行LC的老年患者60例。随机分成对照组和BIS组,每组30例。对照组患者由同一位麻醉医师根据其临床经验调整麻醉深度。BIS组患者根据BIS值调整麻醉深度,BIS维持40～60。观察两组苏醒质量、血流动力学、应激反应和术后认知功能,记录不良反应发生情况。结果：BIS组的苏醒时间、呼吸恢复时间、拔管时间短于对照组（P<0.05）。插入喉罩后1 min（T1）~拔除喉罩后1 min（T3）时间点,BIS组的皮质醇（COR）、血糖低于对照组（P<0.05）。T0~T3时间点,BIS组的平均动脉压（MAP）和心率（HR）低于对照组（P<0.05）。BIS组术后1 d、术后3 d简易精神状态评价量表（MMSE）评分高于对照组（P<0.05）。两组不良反应发生率对比无统计学差异（P>0.05）。结论：BIS监测应用于七氟醚吸入全身麻醉的老年LC患者,可稳定血流动力学,减轻应激反应,提高患者苏醒质量,减轻认知功能损害。     

钙离子浓度对脂肪干细胞增殖和成肌分化的影响

摘要 目的：本研究旨在探讨钙离子浓度对脂肪干细胞（ADSCs）增殖和成肌分化的影响。方法：利用抽脂手术废弃脂肪提取脂肪干细胞,进行体外细胞培养和鉴定。设立正常细胞培养液为对照组,在培养液中添加不同浓度的钙离子（分别为0.001 mol/mL、0.002 mol/mL、0.003 mol/mL、0.004 mol/mL、0.005 mol/mL共5组）为实验组,测定在48 h和96 h条件下不同浓度钙离子(Ca²⁺)对cck8反应细胞增殖的影响。同时通过TGF-β诱导成肌分化,测定不同钙离子浓度下Ca²⁺信号相关蛋白的表达,并通过CCK-8检测评价其分化水平。另外通过免疫荧光和PCR检测细胞表面特异性抗原(α-SMA,SMMHC)的表达水平。各组间比较应用独立样本 t 检验,P<0.05为差异具有统计学意义。结果：显微镜下观察可见原代ADSCs细胞培养0.5 天后贴壁生长,形态呈圆或椭圆形,而TGF-β诱导ADSCs成肌分化可见细胞呈梭形;实验组中,钙浓度<0.002 mmol/mL时,脂肪干细胞增殖得到显著促进（t=10.08, df=10, P<0.05）;钙浓度<0.003 mmol/mL时,脂肪干细胞的成肌分化也显著增加,同时免疫荧光和PCR检测发现低浓度下α-SMA,SMMHC的表达均显著增加（P<0.05）,而高钙浓度下两者均受到抑制（P<0.05）。结论：低钙浓度(钙浓度<0.002 mmol/mL)可显著促进脂肪干细胞增殖和成肌分化,而高钙浓度下两者均受到抑制。     

河北省乡镇卫生院招聘执业医师项目调查与分析

目的 了解河北省乡镇卫生院招聘执业医师项目的实施现状及问题,探索农村卫生机构吸引、稳定人才的长效机制。方法 对河北省张家口等市开展的乡镇卫生院招聘执业医师项目进行调研,主要通过调阅文件资料、个人深入访谈等方式收集资料。结果 受聘执业医师的各项政策基本得到了落实,受聘执业医师在卫生院发挥了重要作用,各方对执业医师评价较高。结论 为改善项目效果,应加大项目的督导力度、为受聘执业医师制定系统的培训计划并逐步提高待遇等。     

喙尾琵琶甲肠道来源链霉菌(Streptomyces sp.)BPA71次级代谢产物的分离鉴定及其生物活性评价

【背景】昆虫是世界上种类最多、肠道菌群资源最丰富且多样的动物类群之一。昆虫肠道微生物具有产生活性次级代谢产物的能力,是活性天然产物的重要来源。【目的】研究药用昆虫喙尾琵琶甲(Blaps rynchopetera)成虫肠道来源链霉菌(Streptomyces sp.) BPA71的次级代谢产物及其生物活性。【方法】利用正相硅胶柱色谱、葡聚糖凝胶Sephadex LH-20柱色谱等方法分离纯化该菌株的发酵粗提物,采用牛津杯法进行抗菌活性追踪,确定抗菌活性部位,通过ESI-MS、NMR等波谱数据分析对化合物结构进行鉴定,采用微量肉汤稀释法测定最低抑菌浓度(minimal inhibitory concentration, MIC),采用MTS法测定抗肿瘤活性。【结果】从Streptomyces sp. BPA71的固体发酵提取物中共分离得到4个已知化合物,通过对比核磁数据确定为糠酸甲酯(1)、吡咯甲酰胺A (2)、吡咯甲酰胺B (3)和吲哚-3-乙酸甲酯(4)。抗菌活性结果显示化合物2具有广谱抗菌活性。此外,化合物2对宫颈癌细胞HeLa、肺癌细胞A549、肝癌细胞SMMC-7721、乳腺癌细胞MDA-MB-231和结肠癌细胞SW480这5株肿瘤细胞均有明显的抑制活性。【结论】喙尾琵琶甲肠道来源Streptomyces sp. BPA71可产生丰富的生物活性物质,该研究结果为进一步挖掘喙尾琵琶甲肠道链霉菌的活性天然产物奠定了基础,同时丰富了人们对喙尾琵琶甲肠道微生物的认识。     

基于PI3K/AKT通路探究上调miR-210对牙髓干细胞增殖、凋亡能力的影响

摘要 目的：研究基于磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路探究上调微小RNA 210(miR-210)对大鼠牙髓干细胞增殖、凋亡能力的影响。方法：选取10只健康Sprague-Dawley（SD）雄性大鼠,颈椎脱臼处死后提取大鼠下切牙牙髓,进行牙髓干细胞培养和鉴定。分为正常组（未进行处理）,miR-210抑制组（给予20 nmol/L的miR-210抑制物）,miR-210对照组（给予20 nmol/L的miR-210模拟物）三组。采用CCK-8法检测牙髓干细胞增殖活性,酶联免疫吸附试验（ELISA）检测ALP活性,流式细胞仪检测细胞凋亡,采用免疫印迹（Western blot）检测PI3K、AKT蛋白。结果：与正常组相比,miR-210抑制组细胞增殖、ALP活性降低,细胞凋亡率升高;miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与miR-210抑制组相比,miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与正常组相比,miR-210抑制组PI3K、p-AKT蛋白表达降低,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。与miR-210抑制组相比,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。结论：miR-210通过调控PI3K、p-AKT蛋白激活PI3K/AKT通路,促进大鼠牙髓干细胞增殖,抑制牙髓干细胞凋亡。     

Normal Values for Segmental Bioimpedance Spectroscopy in Pediatric Patients

Introduction

Localized limb edema is a clinically relevant sign in diseases such as post-thrombotic syndrome and lymphedema. Quantitative evaluation of localized edema in children is mainly done by measuring the absolute difference in limb circumference, which includes fat and fat-free mass. Bioimpedance spectroscopy (BIS) provides information on the fluid volume of a body segment. Our objective was to determine normal ranges for segmental (arm and leg) BIS measurements in healthy children. Additionally, we determined the normal ranges for the difference in arm and ankle circumference and explored the influence of handedness and the correlation between techniques.

Methods

Healthy children aged 1-18 years were recruited. The ratio of extracellular fluid content between contralateral limbs (estimated as the inter-arm and inter-leg extracellular impedance ratio), and the ratio of extracellular to intracellular fluid content for each limb (estimated as the intracellular to extracellular impedance ratio) were determined with a bioimpedance spectrometer. Arm and ankle circumference was determined with a Gulick II tape.

Results

We recruited 223 healthy children (48 infants, 54 preschoolers, 66 school-aged children, and 55 teenagers). Normal values for arm and leg BIS measurements, and for the difference in arm and ankle circumference were estimated for each age category. No influence of handedness was found. We found a statistically significant correlation between extracellular impedance ratio and circumference difference for arms among teenagers.

Conclusion

We determined normal BIS ranges for arms and legs and for the difference in circumference between arms and between ankles in children. There was no statistically significant correlation between extracellular impedance ratio and difference in circumference, except in the case of arms in adolescents. This may indicate that limb circumference measures quantities other than fluid, challenging the adequacy of this technique to determine the presence of localized edema in most age groups.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Electrical impedance measurements in the arm and the leg during a thirty day bed rest study

The need to detect, follow and understand the effects of gravity on body fluid distribution is a constant stimulus to the quest for new techniques in this area of research. One of these techniques is electrical bioimpedance spectroscopy (BIS). Although not new, this is a technique whose applications to biomedical research are fairly recent. What is new is the development of instrumentation that has made practical the use of impedance spectroscopy in the biomedical setting, particularly in studies involving human subjects. The purpose of this paper is to report impedance spectroscopy observations made on a subject who was submitted to bed rest for a period of thirty days. These observations were made as part of a study on muscle atrophy during a thirty day head down bed rest. Since bed rest studies are very costly in human and financial terms, and technically difficult to realize, we felt that even though the present study deals only with a single case it was worthy of reporting because it illustrates kinds of questions impedance spectroscopy may help to answer in microgravity research.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.     

ABCA2基因mRNA在结核分枝杆菌不同感染阶段的表达及其作为诊断标志物的研究

摘要 目的：比较ATP结合转运蛋白A2（ATP-binding cassette transporterA2,ABCA2）基因mRNA在活动性结核病和结核分枝杆菌潜伏感染状态下的表达差异及其作为诊断标志物的能力。方法：收集活动性结核病患者、结核分枝杆菌潜伏感染者和健康人外周血,荧光定量PCR检测受试者外周血单个核细胞中ABCA2的mRNA水平,统计学分析各组ABCA2基因表达差异,及其鉴别活动性结核和潜伏感染的能力。结果：活动性结核病患者外周血单个核细胞中ABCA2的mRNA表达显著低于潜伏感染者和健康人（（H=83.38,P＜0.0001）。ABCA2鉴别诊断活动性结核和潜伏感染的曲线下面积为0.9235,灵敏度为73.53%（95%的置信区间为63.87%~81.78%）,特异性为93.55%（95%的置信区间为78.58%~99.21%）。结论：外周血单个核细胞中ABCA2基因mRNA是鉴别活动性结核病和潜伏感染者的潜在标志物,有助于活动性结核病的辅助诊断。     

Human hydrometry: comparison of multifrequency bioelectrical impedance with 2H2O and bromine dilution

The traditionalmethod of assessing total body water (TBW), extracellular water (ECW),and intracellular water (ICW) has been the use of isotopes, on thebasis of the dilution principle. Although the development ofbioelectrical impedance techniques has eliminated many of themeasurement constraints associated with the dilution methods, thedegree of interchangeability between the two methods remains uncertain.We used multifrequency bioelectrical impedance spectroscopy (BIS),²H₂Odilution, and bromine dilution to assess TBW, ECW, and ICW in 469 healthy subjects (248 males, 221 females) aged 3-29 yr. We foundthat the TBW, ECW, and ICW estimates for the BIS and dilution methodswere significantly correlated(r² = 0.80-0.96, P < 0.0001, SE ofthe estimate = 2.3-2.7 liters). On the basis of population, theconstants used in the BIS analysis could be adjusted so that the meandifferences with the dilution methods would become zero. The SD valuesfor the mean differences between the dilution and BIS methods, however,remained significant for both males and females: TBW (±2.1-2.8liters), ECW (±1.4-1.6 liters), and ICW (2.0-3.1 liters).To improve the accuracy of the BIS measurement for an individual withinthe age range we have examined, further refinement of the constantsused in the BIS analysis is needed.

     

电阻抗法在植物根系生物学研究中的应用

因受限于检测方法,对埋藏于土壤中植物根系研究的深入程度远低于其地上部分。传统获取根系的方法不仅费时费力,而且对根系原位分布和生存微环境具有明显的扰动破坏,故随着根系研究的不断深入,迫切需要寻求一种非破坏性的根系检测方法。电阻抗法是在一定频率的外加电源下,测量电路中的根系电学特征(电容、电阻和电阻抗图谱),并且电学特征与根系生物量和形态指标之间存在较好的相关性,然而,由于对电阻抗法基于电路中电流流向等的关键机理尚不清楚,有些学者对该方法的大范围应用提出了质疑。本文首先对生物电阻抗法测量根系的原理和模型进行阐述,然后重点综述根系电阻抗法研究的不同理论方法及存在的问题,最后提出电阻抗法研究植物根系应该解决的重要科学问题,并展望电阻抗法更加广泛的研究和应用前景,以期为获取非破坏性的根系研究技术提供参考。     

Reliability of bioimpedance spectroscopy and tonometry after breast conserving cancer treatment

BACKGROUND: Measuring the female breast, especially after breast cancer treatment, is problematic due to breast size, texture, and patient positioning. However, being able to accurately measure changes in the breast is important, as it may help in the earlier diagnosis and treatment of early breast edema and later lymphedema. METHODS: 14 women who had undergone breast conserving surgery for breast cancer (> 12 months ago) were recruited to assess the between subject reproducibility of tonometry and bioimpedance spectroscopy (BIS). With the participant supine, two repeat measurements of the resistance of the tissues to compression (tonometry) and fluid levels (BIS) of the treated and normal breast were taken for each of the four quadrants of the breast. RESULTS: The between subject reproducibility for both measurement techniques was high, with covariance ranging from 1.29% to 3.25% for tonometry and 0.20-0.86% for BIS. CONCLUSIONS: The reliability of these two measurement techniques provides an opportunity for researchers and clinicians to easily quantify breast tissue and fluid changes which in turn may lead to the earlier diagnosis and targeted treatment of breast edema and lymphedema.     

一种非酒精性脂肪性肝病的超声影像人工智能辅助诊断系统开发

摘要 目的：结合人工智能方法设计针对肝脏超声影像的辅助诊断系统，辅助医生对大样本肝脏超声影像数据的标准化和高效化诊断，实现基于肝脏超声图像的非酒精性脂肪性肝病的精准诊断。方法：通过开发肝脏超声影像的识别与分类、脂肪肝分级分析和肝脏脂肪含量定量分析三个模块，建立一套非酒精性脂肪性肝病的超声影像人工智能辅助诊断系统，该系统能够自动区分输入到系统中不同采样视野的超声影像类型，并对肝脏超声图像进行数字化分析，给出待测超声图像是否呈现脂肪肝以及其肝脏脂肪含量的百分比值。结果：本研究中的超声图像识别分类模块可高通量区分出肝肾比图像和衰减率图像的两类超声影像，其分类的准确率达100%。脂肪肝分级分析模块在测试集数据的准确率达到84%，展现出可胜任辅助医生诊断的能力。基于人工肝脏脂肪含量定量方法开发的肝脏脂肪含量定量分析模块的准确率达到67.74%。结论：本研究已开发出一套基于肝脏超声影像的智能辅助诊断系统，可以辅助医生快速、简单、无创地筛选出潜在患有脂肪肝的患者，虽然现阶段实现肝脏脂肪定量分析仍有难度，但已展现出较大的临床应用潜力。     

The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.