Leptomeningeal Carcinomatosis: A Call for Optimizing Diagnostic Sensitivity by the Hematology Laboratory

In the cerebrospinal fluid (CSF), the demonstration of malignant cells by cytological examination is currently the gold standard for the diagnosis of leptomeningeal carcinomatosis (LC). However, a positive cytology is observed in only 50–60% of patients with LC and highly dependent on pre-analytical factors. The hematology laboratory could provide an immediate and accurate diagnosis, but diagnostic sensitivity is not always optimized once the sample is received. We hereby report a 49-year-old woman with a 3-year grade III invasive ductal carcinoma who was admitted to the emergency department due to headaches, nausea, and vomiting. The CSF revealed pleocytosis with suspicious high fluorescent cells on the hematology analyzer concomitantly with biochemical alterations. Cytomorphological examination confirmed tumor cells, thus diagnosing a leptomeningeal metastasis of her breast cancer. The patient was eventually transferred to palliative care. Cytological examination is a valuable tool for a rapid diagnosis of LC if diagnostic performance is optimized. In addition to repeated CSF collections with a sufficient volume (5–10 mL), this could be reached by processing the CSF as soon as possible, taking into account the fluorescence information from the analyzer, proceeding systematically to microscopic examination even with normal CSF white blood cell count, and providing quality improvement of the staff to identify malignant cells.

Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum

Background The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Results In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E.limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. Conclusions The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E.limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

High-content imaging-based pooled CRISPR screens in mammalian cells

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeats) -based gene inactivation provides a powerful means of linking genes to particular cellular phenotypes. CRISPR-based screening has mostly relied upon using large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to computationally select cells displaying a particular CRISPR-induced phenotype, mark them by photo-conversion of an expressed photo-activatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photo-conversion of cells, allowing ~1.5 million individual cells to be screened in 8 hr. We used this approach to screen 6092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a highly scalable approach to facilitate imaging-based pooled CRISPR screens.

Изучение регенеративного потенциала стволовых клеток цельного костного мозга для лечения механических травм кожи в модельных экспериментах на мышах

Введение. Культивированные мезенхимальные стромальные стволовые клетки (МССК), выделенные из костного мозга (КМ), можно использовать для лечения обширных ран, однако это очень дорого, трудоёмко и возможно только через несколько дней после их получения. При этом цельный донорский КМ можно вводить системно непосредственно после травмы без выделения МССК, так как они содержатся в нём естественным образом. Это важно для предотвращения гибели и инвалидизации пострадавших. Цель исследования - изучение терапевтического потенциала цельного донорского КМ, пересаживаемого мышам-реципиентам после нанесения им механических травм. Методика. Эксперимент выполнен на 38 животных. На следующий день после облучения в дозе 6,5 Гр 18 мышам-реципиентам линии C57BL/6 наносили резаную рану в межлопаточной области спины, а затем вводили внутривенно 100 мкл суспензии клеток донорского цельного КМ, несущих маркерный ген зеленого флуоресцентного белка EGFP. Реципиентов забивали через 1, 2, 3, 7, 11, 14, 21, 28 и 35 сут после трансплантации. Под флуоресцентным микроскопом изучали участки кожи, прилегающие к ране, а также дно раны и струп. Скорость заселения этих зон сравнивали со скоростью заселения участков кожи без раны на пояснице данных реципиентов и в межлопаточной и поясничной областях (20) у облучённых животных-реципиентов без травмы. Результаты. Уже на следующий день после трансплантации в участках кожи, прилегающих к ране, и на дне раны обнаруживали донорские клетки. Через 7 сут наблюдалось массированное заселение раны флуоресцирующими клетками различных типов; в то же время в участках кожи без раны на пояснице данных реципиентов донорские клетки появились в существенных количествах только через 11 сут. Донорские клетки сохранялись в коже по меньшей мере 35 сут после трансплантации без всяких признаков элиминирования. У животных без травмы заселение кожи донорскими клетками происходило медленнее, чем у травмированных, с похожим типом заселения обеих изучаемых зон (межлопаточной и поясничной). Заключение. Полученные результаты позволяют предположить, что повреждённые ткани выделяют цитокины, обладающие способностью привлекать большинство донорских клеток именно к ране. МССК цельного КМ заращивали рану с очень большой скоростью, из чего можно предположить, что его трансплантация сразу после получения различных травм по эффективности может быть не хуже, чем лечение культивированными МССК, а по оперативности воздействия и экономичности намного его превосходить.Introduction. Cultured mesenchymal stromal stem cells (MSSC), isolated from the bone marrow (BM) may be used to treat extensive wounds, but this treatment is very expensive, time consuming and possible only in several days after the injury. However, donor whole BM can be systemically administered directly after an injury without isolating MSSC because they are naturally contained in the BM. This is important for preventing death and disability of accident victims. The aim of the work was to study the therapeutic potential of donor whole BM transplanted to recipient mice after inflicting a mechanical trauma. Methods. On the next day after irradiation at a dose of 6.5 Gy, recipient C57BL/6 mice were subjected to a cut wound in the interscapulum and then injected intravenously with 100 μl of cell suspension of the donor whole BM carrying a marker gene of the green fluorescent protein, EGFP. Recipients were sacrificed in 1, 2, 3, 7, 11, 14, 21, 28, and 35 days after transplantation. The bottom of the wound and the scab on it and also areas of the skin adjacent to the wound were examined by fluorescent microscopy. The rate of colonization of these zones was compared to the rate of colonization of non-injured lumbar skin areas of these recipients and interscapular and lumbar regions of irradiated recipients without traumas. Results. Already on the next day after transplantation, the donor cells were detected in skin areas adjacent to the wound and on the bottom of the wound. In 7 days, massive wound colonization with various types of fluorescent cells was observed; at the same time, substantial amount of donor cells appeared in the non-injured lumbar skin of these recipients only in 11 days. The donor cells remained in the skin for at least 35 days after transplantation without any signs of elimination. Colonization of skin with the donor cells was slower in animals without than with wounds with a similar type of colonization in both of the studied zones (interscapular and lumbar). Conclusions. The study results suggested that damaged tissues secrete cytokines that are capable of attracting the majority of donor cells specifically to the wound. MSSC of the whole BM healed the wound very fast, which indicated that the MSSC transplantation immediately after a trauma is not inferior in effectiveness to the treatment with cultured MSSC and may be much superior in both promptness of the effect and cost-efficiency.