Live cell pool and rare cell isolation using Enrich TROVO system.

Although several technologies have been developed to isolate cells of interest from a heterogenous sample, clogging and impaired cell viability limit such isolation. We have developed the Enrich TROVO system as a novel, nonfluidic technology to sort live cells. The TROVO system combines imaging-based cell selection and photo-crosslinking of (gelatin methacrylate) gelMA-hydrogel to capture cells. After capture, cells are released by enzymatic digestion of the hydrogel and then retrieved for downstream analysis or further cell culturing. The system can capture cells with a recovery rate of 48% while maintaining 90% viability. Moreover, TROVO can enrich rare cells 506-fold with 93% efficiency using single step isolation from a 1:104 cell mixture, and can also capture one target cell from 1 million cells, reaching an enrichment ratio of 9128. In addition, 100% purity and 49% recovery rate can be achieved by a following negative isolation process. Compared to existing technologies, the TROVO system is clog-resistant, highly biocompatible, and can process a wide range of sample sizes.

Primary breast diffuse large B-cell lymphoma in the rituximab era: A retrospective study of the Chinese Southwest Oncology Group.

BACKGROUND: Primary breast diffuse large B-cell lymphoma (PB-DLBCL) is a rare subtype of extranodal DLBCL, and the standard treatment remains controversial. In this study, we aimed to define the optimal treatment management in the rituximab era. METHODS: A total of 5089 newly diagnosed DLBCL patients treated with rituximab-containing immunochemotherapy between 2008 and 2019 from the Chinese Southwest Oncology Group-affiliated institutes were identified, of whom 135 diagnosed with PB-DLBCL were eligible for this analysis. RESULTS: PB-DLBCL accounted for 2.7% of all DLBCLs. With a median follow-up of 4.2 years, the 5-year overall survival and progression-free survival rates were 84.8% and 71.6%, respectively. Breast and central nervous system (CNS) relapses were the main cause of treatment failure. We observed that consolidative breast radiotherapy (RT) significantly decreased breast relapse risk (5-year risk, 2.9% vs. 20.1%, p = 0.007). The CNS relapse risk was lower for patients who received high-dose methotrexate (HD-MTX) than for patients who did not (5-year risk, 0% vs. 15.2%, p = 0.015). We further screened the genetic mutation profile of 20 patients from two institutes, and found that MYD88 (25%) and CD79B mutations (25%) frequently occur in PB-DLBCL. In addition, four patients with MYD88 and/or CD79B mutations experienced CNS relapse, while three patients with MYD88 and/or CD79B mutations who received HD-MTX did not experience CNS relapse. CONCLUSION: Collectively, our results indicate combined modality therapy including rituximab-containing immunochemotherapy and consolidative breast RT is a promising approach for PB-DLBCL, while HD-MTX is useful for preventing CNS relapse.

Prevalence of acute myeloid leukemia and its associated risk factors at a tertiary care center: a retrospective cross-sectional study.

Background: Acute myeloid leukemia (AML) is a common malignant disorder of the bone marrow, accounting for 23.1% of total leukemia cases globally in 2017. This study aimed to know the prevalence of AML in a tertiary hospital center in Nepal and assess the associated etiological factors in the causation of AML. Materials and methods: A hospital-based retrospective cross-sectional study was conducted among patients with acute leukemia admitted to a tertiary care center in Kathmandu from 1 April 2021 to 30 April 2022. Three hundred and ten participants with acute leukemia were selected via a convenience sampling technique. The data were entered into Microsoft Excel 365 and then analyzed using Statistical Packages for Social Sciences version 22.0. Results: One hundred and forty five (46.7%) out of 310 acute leukemia patients had AML. Most of the cases were male (104, 71.7%) and aged 40-49 years (36, 24.8%). The most common signs and symptoms were pallor (115, 79.3%), fever (n=92, 63.4%), and lethargy/weakness (78, 53.8%). Ninety one (62.8%) patients underwent multiple cycles of chemotherapy while 51 (35.2%) had supportive care only, and 3 (2.1%) went bone marrow transplants. Conclusion: The prevalence of AML was higher as compared to AML in other studies done in a similar setting. Early diagnosis and treatment are key to a good prognosis and cure in most cases.

Selective expansion of target cells using the Enrich TROVO platform.

Enriching target cell clones from diverse cell populations is vital for many life science applications. We have developed a novel method to rapidly and efficiently purify specific clonal cell populations from a larger, heterogeneous group using the Enrich TroVo system (Enrich Biosystems Inc., CT, USA). This system takes advantage of microfabrication and optical technologies by utilizing small hydrogel wells to separate desired cell populations and an innovative patching technique to selectively eliminate undesired cells. This method allows the isolation and growth of desired cells with minimal impact on their viability and proliferation. We successfully isolated and expanded clonal cell populations of desired cells using two model cells. Compared with fluorescence-activated cell sorting, Enrich TroVo system offers a promising alternative for isolating of sensitive, adherent cells, that is, patient-derived cells.

Efficient end-to-end learning for cell segmentation with machine generated weak annotations.

Automated cell segmentation from optical microscopy images is usually the first step in the pipeline of single-cell analysis. Recently, deep-learning based algorithms have shown superior performances for the cell segmentation tasks. However, a disadvantage of deep-learning is the requirement for a large amount of fully annotated training data, which is costly to generate. Weakly-supervised and self-supervised learning is an active research area, but often the model accuracy is inversely correlated with the amount of annotation information provided. Here we focus on a specific subtype of weak annotations, which can be generated programmably from experimental data, thus allowing for more annotation information content without sacrificing the annotation speed. We designed a new model architecture for end-to-end training using such incomplete annotations. We have benchmarked our method on a variety of publicly available datasets, covering both fluorescence and bright-field imaging modality. We additionally tested our method on a microscopy dataset generated by us, using machine-generated annotations. The results demonstrated that our models trained under weak supervision can achieve segmentation accuracy competitive to, and in some cases, surpassing, state-of-the-art models trained under full supervision. Therefore, our method can be a practical alternative to the established full-supervision methods.

Hematopoietic Stem Cell Transplantation in Nepal: International Partnership, Implementation Steps, and Clinical Outcomes.

Blood and marrow transplantation (BMT) is rarely available in many low- to middle-income countries (LMICs). In 2012, Civil Service Hospital, a government hospital in Kathmandu, partnered with the University of Illinois at Chicago to consult on the establishment of BMT in their hospital, train staff, and promote educational activities. The implementation of BMT occurred in 3 phases over 4 years and included regular onsite visits, training of personnel in Chicago, continuous remote communication, and co-organization of educational events in Kathmandu. The Nepalese government funded the construction of a state-of-the art BMT unit and stem cell laboratory inside Civil Hospital. Autologous (auto) hematopoietic stem cell transplantation (HSCT) was started in 2016, and allogeneic (allo) HSCT from matched related donors (MRDs) or haploidentical (haplo) donors was initiated in 2017. The cost of transplantation was $5200 for auto-HSCT, $10,000 for MRD HSCT, and $13,300 for haplo HSCT. The major socioeconomic determinants reported by Nepalese BMT providers were the cost of transplantation, loss of revenue of the patient and/or caregiver, and cost of transportation. All patients (n = 66) received peripheral blood stem cell grafts, and all allo-HSCT recipients were given post-transplantation cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis. Among recipients of auto-HSCT (n = 30), with a median follow-up of 1029 days (range, 130 to 1653 days), 87% were alive, and transplantation-related mortality (TRM) was 10%. Among allo-HSCT recipients (n = 36), all patients engrafted, and at a median follow-up of 204 days (range, 12 to 1131 days), 75% of them were alive (MRD, 71%; haplo, 83%), with a TRM of 19%. Only 3 of 36 patients developed acute GVHD grade II-IV. The median overall survival in auto-HSCT recipients was 1610 days and was not reached in allo-HSCT recipients. The long-lasting partnership with University of Illinois at Chicago helped build capacity and allowed the Civil Service Hospital team to establish a BMT program in Nepal that has high quality standards at an affordable cost for the majority of patients.

Synthesis and Spectral Studies of Some Homo Dinuclear Lanthanide(III) Complexes of a Mesogenic Schiff Base.

A mesogenic Schiff base, N,N'-di(4-decyloxysalicylidene)-1',8'-diamino-3',6'-dioxaoctane (H2L) and a series of homo dinuclear lanthanide(III) complexes of the type [Ln2(LH2)3(NO3)4](NO3)2, (Ln = La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, and Ho) were synthesized and characterized by elemental analysis, mass spectrometry, FTIR, and NMR spectral techniques. The IR and NMR spectral evidences imply bonding of a neutral bidentate H2L species through two phenolate oxygen atoms in its zwitterionic form to LnIII, rendering the overall geometry of the complexes as a seven-coordinate polyhedron - possibly distorted mono-capped octahedron. Differential scanning calorimetry (DSC) and polarizing optical microscopic (POM) studies reveal mesogenic properties (smectic-X, smectic-A and nematic mesophases) in the ligand over a wide range of temperature but none mesomorphism in the LnIII complexes synthesized under this study. Luminescence studies exhibit emissions of H2L and TbIII complex.

A highly selective pyridoxal-based chemosensor for the detection of Zn(ii) and application in live-cell imaging; X-ray crystallography of pyridoxal-TRIS Schiff-base Zn(ii) and Cu(ii) complexes.

In a simple, one-step reaction, we have synthesized a pyridoxal-based chemosensor by reacting tris(hydroxymethyl)aminomethane (TRIS) together with pyridoxal hydrochloride to yield a Schiff-base ligand that is highly selective for the detection of Zn(ii) ion. Both the ligand and the Zn(ii) complex have been characterized by 1H & 13C NMR, ESI-MS, CHN analyses, and X-ray crystallography. The optical properties of the synthesized ligand were investigated in an aqueous buffer solution and found to be highly selective and sensitive toward Zn(ii) ion through a fluorescence turn-on response. The competition studies reveal the response for zinc ion is unaffected by all alkali and alkaline earth metals; and suppressed by Cu(ii) ion. The ligand itself shows a weak fluorescence intensity (quantum yield, Φ = 0.04), and the addition of zinc ion enhanced the fluorescence intensity 12-fold (quantum yield, Φ = 0.48). The detection limit for zinc ion was 2.77 × 10-8 M, which is significantly lower than the WHO's guideline (76.5 µM). Addition of EDTA to a solution containing the ligand-Zn(ii) complex quenched the fluorescence, indicating the reversibility of Zn(ii) binding. Stoichiometric studies indicated the formation of a 2 : 1 L2Zn complex with a binding constant of 1.2 × 109 M-2 (±25%). The crystal structure of the zinc complex shows the same hydrated L2Zn complex, with Zn(ii) ion binding with an octahedral coordination geometry. We also synthesized the copper(ii) complex of the ligand, and the crystal structure showed the formation of a 1 : 1 adduct, revealing 1-dimensional polymeric networks with octahedral coordinated Cu(ii). The ligand was employed as a sensor to detect zinc ion in HEK293 cell lines derived from human embryonic kidney cells grown in tissue culture which showed strong luminescence in the presence of Zn(ii). We believe that the outstanding turn-on response, sensitivity, selectivity, lower detection limit, and reversibility toward zinc ion will find further application in chemical and biological science.

Effects of estradiol on lactoprivic signaling of the hindbrain upon the contraregulatory hormonal response and metabolic neuropeptide synthesis in hypoglycemic female rats.

BACKGROUND: Caudal dorsomedial hindbrain detection of hypoglycemia-associated lactoprivation regulates glucose counter-regulation in male rats. In females, estradiol (E) determines hypothalamic neuroanatomical and molecular foci of hindbrain energy sensor activation. This study investigated the hypothesis that E signal strength governs metabolic neuropeptide and counter-regulatory hormone responses to hindbrain lactoprivic stimuli in hypoglycemic female rats. METHODS: Ovariectomized animals were implanted with E-filled silastic capsules [30 (E-30) or 300 µg (E-300)/mL] to replicate plasma concentrations at estrous cycle nadir versus peak levels. E-30 and E-300 rats were injected with insulin or vehicle following initiation of continuous caudal fourth ventricular L-lactate infusion. RESULTS: Hypoglycemic hypercorticosteronemia was greater in E-30 versus E-300 animals. Glucagon and corticosterone outflow was correspondingly fully or partially reversed by hindbrain lactate infusion. Insulin-injected rats exhibited lactate-reversible augmentation of norepinephrine (NE) accumulation in all preoptic/hypothalamic structures examined, excluding the dorsomedial hypothalamic nucleus (DMH) where hindbrain lactate infusion either suppressed (E-30) or enhanced (E-300) NE content. Expression profiles of hypoglycemia-reactive metabolic neuropeptides were normalized (with greater efficacy in E-300 animals) by lactate infusion. DMH RFamide-related peptide-1 and -3, arcuate neuropeptide Y and kisspeptin, and ventromedial nucleus nitric oxide synthase protein responses to hypoglycemia were E dosage-dependent. CONCLUSIONS: Distinct physiological patterns of E secretion characteristic of the female rat estrous cycle elicit differential corticosterone outflow during hypoglycemia, and establish both common and different hypothalamic metabolic neurotransmitter targets of hindbrain lactate deficit signaling. Outcomes emphasize a need for insight on systems-level organization, interaction, and involvement of E signal strength-sensitive neuropeptides in counter-regulatory functions.

Hindbrain 5'-Adenosine Monophosphate-activated Protein Kinase Mediates Short-term Food Deprivation Inhibition of the Gonadotropin-releasing Hormone-Luteinizing Hormone Axis: Role of Nitric Oxide.

Hindbrain-derived stimuli restrain the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) reproductive neuroendocrine axis during energy insufficiency. Interruption of food intake, planned or unplanned, is emblematic of modern life. This study investigated the premise that the hindbrain energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibits reproductive neuroendocrine function in short term, e.g. 18-h food-deprived (FD) estradiol (E)-implanted ovariectomized female rats. Intra-caudal fourth ventricular administration of the AMPK inhibitor Compound C (Cc) reversed FD-induced inhibition of rostral preoptic (rPO) GnRH protein expression and LH release in animals given E to replicate proestrus (high-E dose-, but not metestrus (low-E dose)-stage plasma steroid levels. FD caused Cc-reversible augmentation or diminution of preoptic norepinephrine (NE) activity in high- versus low-E rats, respectively, and AMPK-independent reductions in hypothalamic NE accumulation in the latter. Nitric oxide (NO) and kisspeptin are key stimulatory signals for the preovulatory LH surge. Here, FD inhibited rPO neuronal nitric oxide synthase protein expression in high-, but not low-E-dosed animals. Lateral ventricular delivery of the NO donor 3-morpholinosydnonimine (SIN-1) reversed inhibitory GnRH and LH responses to FD in high-E rats, and normalized rPO Vglut2, anteroventral periventricular KiSS1, and dorsomedial hypothalamic RFRP-3 mRNA and/or protein profiles. Data show that FD curtails reproductive neuroendocrine outflow by hindbrain AMPK-dependent mechanisms in the presence of peak estrous cycle E levels. Results indicate that neural networks linking this sensor to GnRH neurons likely involve NO signaling, which may function upstream of one or more neurotransmitters identified here by SIN-1-reversible inhibitory responses to FD.

Role of hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) in hypothalamic AMPK and metabolic neuropeptide adaptation to recurring insulin-induced hypoglycemia in the male rat.

Glucose counter-regulatory dysfunction correlates with impaired activation of the hypothalamic metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK). Hypothalamic AMPK is controlled by hindbrain energy status; we examined here whether hindbrain AMPK regulates hypothalamic AMPK and metabolic neurotransmitter maladaptation to recurring insulin-induced hypoglycemia (RIIH). Brain tissue was harvested after single versus serial insulin (I) dosing for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant biosynthetic enzyme/neuropeptide expression in micro-punch dissected arcuate (ARH), ventromedial (VMH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissue. The AMPK inhibitor compound c (Cc) or vehicle was administered to the caudal fourth ventricle ahead of antecedent I injections. RIIH caused site-specific elevation (ARH, VMH, LHA) or reduction (DMH) of total AMPK protein versus acute hypoglycemia; Cc respectively exacerbated or attenuated this response in the ARH and VMH. Hindbrain AMPK correspondingly inhibited or stimulated LHA and DMH pAMPK expression during RIIH. RIIH elicited Cc-reversible augmentation of VMH glutamate decarboxylase profiles, but stimulated (ARH pro-opiomelanocortin; LHA orexin-A) or decreased (VMH nitric oxide synthase) other metabolic neurotransmitters without hindbrain sensor involvement. Results demonstrate acclimated up-regulation of total AMPK protein expression in multiple hypothalamic loci during RIIH, and document hindbrain sensor contribution to amplification of this protein profile in the VMH. Concurrent lack of net change in ARH and VMH tissue pAMPK implies adaptive reductions in local sensor activity, which may/may not reflect positive gain in energy state. It remains unclear if 'glucose-excited' VMH GABAergic and/or ARH pro-opiomelanocortin neurons exhibit AMPK habituation to RIIH, and whether diminished sensor activation in these and other mediobasal hypothalamic neurotransmitter populations may contribute to HAAF.

Hindbrain estrogen receptor-beta antagonism normalizes reproductive and counter-regulatory hormone secretion in hypoglycemic steroid-primed ovariectomized female rats.

Hindbrain dorsal vagal complex A2 noradrenergic signaling represses the pre-ovulatory luteinizing hormone (LH) surge in response to energy deficiency. Insulin-induced hypoglycemia augments A2 neuron adenosine 5'-monophosphate-activated protein kinase (AMPK) activity and estrogen receptor-beta (ERß) expression, coincident with LH surge suppression. We hypothesized that ERß is critical for hypoglycemia-associated patterns of LH secretion and norepinephrine (NE) activity in key reproduction-relevant forebrain structures. The neural mechanisms responsible for tight coupling of systemic energy balance and procreation remain unclear; here, we investigated whether ERß-dependent hindbrain signals also control glucose counter-regulatory responses to hypoglycemia. Gonadal steroid-primed ovariectomized female rats were pretreated by caudal fourth ventricular administration of the ERß antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) or vehicle before insulin injection at LH surge onset. Western blot analysis of laser-microdissected A2 neurons revealed hypoglycemic intensification of AMPK activity and dopamine-ß-hydroxylase protein expression; the latter response was attenuated by PHTPP pretreatment. PHTPP regularized LH release, but not preoptic GnRH-I precursor protein expression in insulin-injected rats, and reversed hypoglycemic stimulation of glucagon and corticosterone secretion. Hypoglycemia caused PHTPP-reversible changes in NE and prepro-kisspeptin protein content in the hypothalamic arcuate (ARH), but not anteroventral periventricular nucleus. Results provide novel evidence for ERß-dependent caudal hindbrain regulation of LH and counter-regulatory hormone secretion during hypoglycemia. Observed inhibition of LH likely involves mechanisms at the axon terminal that impede GnRH neurotransmission. Data also show that caudal hindbrain ERß exerts site-specific control of NE activity in forebrain projection sites during hypoglycemia, including the ARH where prepro-kisspeptin may be a target of that signaling.

Sex-specific basal and hypoglycemic patterns of in vivo caudal dorsal vagal complex astrocyte glycogen metabolic enzyme protein expression.

Astrocytes contribute to neurometabolic stability through uptake, catabolism, and storage of glucose. These cells maintain the major brain glycogen reservoir, which is a critical fuel supply to neurons during glucose deficiency and increased brain activity. We used a combinatory approach incorporating immunocytochemistry, laser microdissection, and Western blotting to investigate the hypothesis of divergent expression of key enzymes regulating glycogen metabolism and glycolysis during in vivo normo- and/or hypoglycemia in male versus female hindbrain astrocytes. Glycogen synthase (GS) and glycogen phosphorylase (GP) levels were both enhanced in dorsal vagal complex astrocytes from vehicle-injected female versus male controls, with incremental increase in GS exceeding GP. Insulin-induced hypoglycemia (IIH) diminished GS and increased glycogen synthase kinase-3-beta (GSK3ß) expression in both sexes, but decreased phosphoprotein phosphatase-1 (PP1) levels only in males. Astrocyte GP content was elevated by IIH in male, but not female rats. Data reveal sex-dependent sensitivity of these enzyme proteins to lactate as caudal hindbrain repletion of this energy substrate fully or incompletely reversed hypoglycemic inhibition of GS and prevented hypoglycemic augmentation of GSK3ß and GP in females and males, respectively. Sex dimorphic patterns of glycogen branching and debranching enzyme protein expression were also observed. Levels of the rate-limiting glycolytic enzyme, phosphofructokinase, were unaffected by IIH with or without lactate repletion. Current data demonstrating sex-dependent basal and hypoglycemic patterns of hindbrain astrocyte glycogen metabolic enzyme expression imply that glycogen volume and turnover during glucose sufficiency and shortage may vary accordingly.

[Pro-apoptotic effect of ciglitazone in leukemic HL-60 cells via PPARγ and P38 MAPK signaling pathway].

OBJECTIVE: To investigate the apoptosis-inducing effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone (CGZ) on leukemic HL-60 cells and its mechanisms of action. METHODS: HL-60 cells in vitro culture medium were subject to different concentrations of CGZ (10-50 µmol/L) for 24, 48 and 72 h. MTT assay was used to detect the cell inhibitory rate and agarose gel electrophoresis to observe DNA fragmentation. Flow cytometry (FCM) and Annexin V/PI staining were used to detect CGZ and/or GW9662 (PPARγ antagonist)-induced cell apoptosis. The expression of PPARγ was examined by RT-PCR and Western blotting. The caspase-3 and protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) were also detected. RESULTS: CGZ (over 30 µmol/L) could inhibit the growth of HL-60 cells in both time- and dose-dependent manner. After treatment for 72 h, the cell growth inhibitory rate in 50 µmol/L CGZ (84% ± 11%) treated cells was found more higher than that in both 40 µmol/L and 30 µmol/L CGZ treated cells (72% ± 13%, 59% ± 13%, P < 0.01) and a typical DNA ladder was also observed by agarose gel electrophoresis. The expression of PPARγ was gradually up-regulated by CGZ treatment and could be down-regulated partially by PPARγ antagonist GW9662. The results also revealed that CGZ-induced cell apoptosis (49.7%, 72 h) could not be inhibited thoroughly by GW9662 (36.2%, control:3.2%). It indicated that the CGZ-induced cell apoptosis was partially PPARγ-independent. Western blotting showed a cleavage of caspase-3 zymogen protein and up-regulation of p-P38 protein. Thus it indicated that the activations of caspase-3 and P38 MAPK were involved in CGZ-induced cell apoptosis. CONCLUSION: CGZ inhibits cell growth by induction of cell apoptosis in HL-60 cells via PPARγ dependent and independent signaling pathways. The activations of caspase-3 and P38 MAPK may be one of the important mechanisms in CGZ in treated HL-60 cells.