H-D-JH rearrangements used by AML-derived Ig were biased in each AML patient. In particularly, the Vκ-Jκ rearrangements were skewed in both AML blasts and normal peripheral blood mononucleated cells (PBMCs). However, AML-derived IGK showed high somatic mutation rates (>2%), while IGK in normal PBMCs rarely displayed hypermutation (<2%). More importantly, we identified five mutation hotspots at serine codons of IGKV3-20 in AML blasts, which may be involved in leukemogenesis and serve as a novel marker for disease monitoring and target therapy.Identifying new molecular targets is of great importance for prognosis prediction and target therapy of acute myeloid leukemia (AML). We previously reported on frequent expression of immunoglobulin (Ig) in myeloblasts. In this study, we investigated the clinical significance of Ig expression in sorted myeloblasts from 59 AML patients. We found that a higher level of AML-derived Ig expression correlated with a significantly shorter disease-free survival. Furthermore, we performed a comprehensive analysis of AML-derived Ig repertoire by next-generation sequencing (NGS) in 16 patients. The transcripts of AML-derived Ig shared some features with B cell-derived Ig, such as a typical V(D)J recombination and high mutation rates. However, they also showed distinct features. In contrast to the huge diversity of classical Ig, the VH-D-JH rearrangements in myeloblasts were biased with individual specificity rather than generally diverse as in B-cells. Compared to AML-derived IgH, AML-derived IGK was more conserved among different AML samples. The frequently shared Vκ-Jκ patterns were IGKV3-20*01/IGKJ1*01, IGKV2D-28*01/IGKJ1*01, and IGKV4-1*01/IGKJ1*01. Moreover, AML-derived IGK was different from classical IGK in B-cells for the high mutation rates and special mutation hotspots at serine codons. Findings of the distinct Ig repertoire in myeloblasts may facilitate the discovery of a new molecular marker for disease monitoring and target therapy.Immunoglobulin (Ig) is known as a hallmark of B-lymphocytes exerting antibody functions. However, our previous studies demonstrated that myeloblasts from acute myeloid leukemia (AML) patients could also express Ig with distinct roles. Here, we quantified Ig (IGHG and IGK) transcripts by real-time PCR and performed a comprehensive analysis of Ig repertoire (both heavy chains and light chains) in AML blasts. We found that Ig was frequently expressed by AML blasts. A higher level of AML-derived IGHG expression correlated with a significantly shorter disease-free survival. Next-generation sequencing revealed dysregulated transcripts of all five Ig classes  and two Ig types (IGK and IGL) in AML. V Acute myeloid leukemia (AML) is a group of genetically heterogeneous diseases characterized by abnormal overproduction of immature myeloid cells, resulting in hematopoiesis impairment and bone marrow failure ,2. Despi+ stem/progenitor cells (IgM) [Monoclonal immunoglobulin (Ig) gene rearrangement has been used as a diagnostic tool for B-cell lymphomas . Althougls (IgM) , AML blals (IgM) ,17,18, als (IgM) ,17,18. Fls (IgM) .Immunoglobulin consists of two identical heavy chains (IgH) and light chains (IgL). There are five classes of IgH, including IgM, IgG, IgA, IgD, and IgE. The two types of IgL are Igκ and Igλ. Each of the IgH and IgL chains contains a variable region and a constant region. The variable (V) region of the IgH gene is formed by combining IGHV, IGHD, and IGHJ genes, whereas the V region of the IgL gene is generated by combining an IGKV with IGKJ gene or an IGLV with IGLJ gene. The constant region  determines the class of IgH. The diversity of Ig results from the V(D)J recombination and random insertions and deletions of non-templated (N) nucleotide at the junctions. Additional diversity is generated by somatic hypermutation (SHM), which is traditionally believed to occur after activation through exposure to an antigen . The mutIn this study, IGHG and IGK expression were quantified in sorted myeloblasts from 59 AML patients by quantitative real-time polymerase chain reaction (qPCR). We assessed mRNA expression levels and correlated them with patients’ clinical features and prognosis. The higher expression level of AML-derived IgG transcripts correlated with shorter disease-free survival (DFS). Since the function of the Ig is highly correlated with its V region characteristics, we performed a comprehensive sequencing analysis of AML-derived Ig repertoire in 16 patients. All five classes of Ig heavy chain and two types of Ig light chain were amplified for each sample by combined multiplex PCR and next-generation sequencing (NGS). Our results demonstrated the expression of Ig with individually biased repertoire and specific SHM hotspots in myeloblasts, opening a potential venue for AML monitoring and precision therapy.Peripheral blood or bone marrow specimens were obtained from AML patients seen at the University of Texas MD Anderson Cancer Center, with written informed consent. The study was conducted according to an Institutional Review Board-approved protocol of the MD Anderson Cancer Center. The clinical data were obtained by review of medical records.For sorting myeloblasts from AML patients, mononuclear cells were isolated from peripheral blood or bone marrow by Ficoll–Hypaque  density gradient centrifugation. The mononuclear cells were stained with two monoclonal antibodies: anti-CD45-APC-Vio770 , and anti-CD19-APC  at 4 °C for 30 min. After washing with PBS (supplemented with 2% FBS) three times, the cells were sorted using a FACScan instrument . We gated on myeloblasts , excluding lymphocytes . We then used CD19 to further exclude B-cell contamination from the blasts A.−ΔΔCt method where ΔCt = Ct(Target) − Ct(Reference). The primers for IGHG constant regions were 5′-ACTACAAGACCACGCCTCC-3′ and 5′-CGTCGCACTCATTTACCC-3′. The primers for IGK constant regions were 5′-CTGTCTTCATCTTCCCGCCA-3′ and 5′-CTTGCTGTCCTGCTCTGTGA-3′.Total RNA was isolated from sorted cells using TRIzol reagent . First-strand cDNA was synthesized from total RNA using oligo dT(15) primers and Moloney murine leukemia virus reverse transcriptase . Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Thermo Fisher) on ABI 7500 System (Thermo Fisher). The expression of the target gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Fold change was calculated by the 2®. During the first round, reverse transcription was completed, and nested gene-specific primers complementary to V and C genes were used to introduce barcodes and sequencing primers into PCR products. The second-round PCR was carried out using communal (sequencing) primers for exponential amplification. Therefore, the entire repertoire was amplified evenly and semi-quantitatively, without introducing additional amplification bias  and IgL  were used to perform two rounds of PCR under the reaction conditions specified by iRepertoireion bias A. The su® company provided the basic data analysis, including barcode de-multiplexing and filtering, V, D, J, C mapping, complementary determining regions (CDR) identification, CDR3 length distribution, and CDR3 algebra (compare repertoires and identify shared CDR3s). The analyzed data in CSV format were then given to us for further analysis using Python and MATLAB programs. Each IgH class or IgL type frequency was calculated based on its reads divided by total reads achieved in the sample. For SHM analysis, filtered DNA sequences were converted to FASTA format and then uploaded to the IMGT/High V-Quest web-based analysis tool [® as the control in data analysis, which was amplified from a mixture of total RNA from the peripheral blood mononucleated cells (PBMCs) of 40 healthy people, with the same primer sets and process flow. Similarly, the normal IGK data was amplified from a mixture of total RNA from PBMCs of 426 healthy people.The iRepertoiresis tool . The anap value of <0.05 was considered statistically significant. Clinicopathologic parameters and gene mutation status between the two groups (high versus low expression) for each immunoglobulin were compared by Pearson’s chi-square test or Fisher exact test.Statistical analyses were performed using GraphPad Prism 6 software and SPSS16. ROC curves were created by plotting sensitivity (Se(c)) and 1-specificity (1-Sp(c)) for all possible threshold values (c) of a marker. The Youden index, where the threshold value for which Se(c) + Sp(c) − 1 is maximized, was then used to provide a criterion for choosing the optimal cutoff value (c*). The cutoff values for immunoglobulin expression were determined by the Youden Index derived from ROC curves. Spearman correlation analysis was used to evaluate correlations among groups assigned, based on two different immunoglobulins . DFS was calculated from the date of initial diagnosis to the date of relapse, death, or last follow-up. DFS was analyzed using the Kaplan–Meier method and compared using the log-rank test. A We have detected Igs on the cell surfaces of myeloblasts by multicolor flow cytometry . High le+ B-cells by fluorescence-activated cell sorting (FACS)  from the 59 patients, excluding lymphocytes , especially CD19g (FACS) A. To assg (FACS) B. We foup = 0.047). No statistically significant differences were noted between the two groups in regards to other clinicopathologic features assessed, including gender, white blood cell (WBC), hemoglobulin level, platelet count, absolute monocyte count, serum level of lactate dehydrogenase (LDH), dysplasia, and WHO classification (p = 0.03). NGS mutation analysis was performed on all patients using a panel of 81 genes that are commonly mutated in hematopoietic neoplasms. The analysis detected at least one gene mutation in 57 (97%) cases. High levels of IgG expression were associated with KIT mutations (p = 0.02) and NPM1 mutations (p = 0.03)  . All tenThe huge diversity is a unique feature of B cell-derived Igs, which function as antibodies against various antigens. Our previous studies have shown that cancer-derived Ig V genes have important tumor biological activities. Since the function of Ig is mainly determined by the characteristics of V region, we explored the signature of AML-derived Ig sequences. We selected myeloblasts from 16 AML patients (10 men and 6 women) and performed a comprehensive sequencing analysis of both IgH and IgL repertoire by combined multiplex PCR A and NGSWe amplified all five classes of heavy chain  and two types of light chain (IGK and IGL) in the blasts from the 16 AML patients. An average of 350,000 Ig heavy chain sequences and 370,000 Ig light chain sequences were obtained for each sample by NGS B. The IGAlthough there are 38 IGKV segments in the human genome, limited segments were used by myeloblasts, as well as normal B cells A. IGKV3-H segments were used by myeloblasts at levels different from that in normal PBMCs. For example, 14 of 16 AML cases used IGHV3-48 at an average usage of 9.4%, much higher than that in normal PBMCs (5.6%). Overall, IGHV1-69, IGHV1-18, IGHV3-11, IGHV2-5, IGHV1-2, and IGHV4-31 were used more frequently in myeloblasts than normal PBMCs , but also in myeloblasts . However, many other Val PBMCs B. Similaal PBMCs D.Theoretically, the 43 IGK genes (38 IGKV and 5 IGKJ) and 73 functional IGH genes  can generate 190 (38 × 5) possible IGK and 6072 (44 × 23 × 6) possible IGH rearrangement patterns, respectively. However, Ig rearrangement patterns used by myeloblasts were biased individually, which means that the blasts from each AML patient had their dominant V(D)J patterns. Interestingly, we found that different patients frequently shared three Vκ-Jκ patterns: IGKV3-20*01/IGKJ1*01 , IGKV2D-28*01/IGKJ1*01 , and IGKV4-1*01/IGKJ1*01  . The preHDJH patterns in myeloblasts also lacked diversity, with the top 1 used VHDJH having usage of over 30% in some patients . However, we failed to identify any overlapping VHDJH rearrangements that were frequently used by different individuals J rearrangement contributes to Ig diversity. The CDR3 of the Ig heavy chain, which falls at the V-D-J joining ends, is the most polymorphic region of Ig. Therefore, Ig diversity correlates with IgH-CDR3 variability . Under pAs known, the Ig gene undergoes SHM, affecting its variable region genes upon antigen stimulation. In B cells, SHM increases the affinity of the antibody. We further explored if SHM also occurred in AML-derived Igs. The mutation status is designated as unmutated if there are <2% mutations and as mutated if there are ≥2% mutations compared to the germline sequences. For Igκ, we analyzed the widely shared IGKV3-20 and IGKV2D-28 as models. Surprisingly, in normal PBMCs, they were both conservative without SHM. However, in AML blasts, IGKV3-20 sequences were mutated in 9/11(82%) samples A. SimilaH usage  varied among patients  sequences: silence mutation (AA not changed), missense mutation (AA changed), and nonsense mutation (stop codon introduced). Most mutations in Vκ were non-silent . We furtWe recently reported that detection of a high level of IgG on AML blasts by flow cytometry analysis, coupled with Igκ or Igλ, is associated with poor overall survival . ConsideOnly IGHG, IGHM, and IGK transcripts were assessed in myeloblasts in previous studies ,17,18. TWe have previously used Sanger sequencing to assess AML-derived Ig rearrangements and found clues for their bias and conservation ,17,18. IIt is known that SHM in B-cells is mediated by AID ,50. WherIn this study, five special mutation hotspots were identified in AML-derived IGKV3-20 . Compared with IGKV2D-28, whose expression and mutation rates varied among different patients, we think that IGKV3-20 may serve as a more suitable target with specific imprints of myeloblasts for tracking AML MRD. More surprisingly, we found that IGKV3-20 mutations preferentially caused replacements of serine (S), which is one of the three AA residues commonly phosphorylated by kinases during cell signaling in eukaryotes. Moreover, it can undergo O-linked glycosylation. Therefore, the replacements of serine may lead to a change in Igκ functions via phosphorylation or O-glycosylation. The main substitutions were Asn (N) and Thr (T). The change to Asn (S30N and S32N), which is very dissimilar to Ser in hydropathy, volume, and chemical characteristics, could bring in classic (N-X-T/S) or non-classic (T/S-X-N) N-glycosylation. It is known that glycosylation is essential for the function of Ig  ,53. We hIn summary, we have found that all the five classes of Ig heavy chains and two types of Ig light chains could be expressed in AML blasts, and that a higher level of AML-derived IgG expression correlated with significantly shorter DFS. AML-derived Igs had biased as well as individualized variable region sequences. Compared to AML-derived IgH, AML-derived IGK was more conserved among different AML samples. Moreover, it differed from classical IGK in B-cells in their hypermutation rate and special mutation hotspots at the serine site. AML-derived Ig, especially Igκ, may play a role as a novel AML-related gene that contributes to leukemogenesis and AML progression. It may also serve as a molecular marker for MRD monitoring, risk stratification, and the development of target therapy.


Our GMN with the distinct intercellular coupling in conjunction with the intracellular feedback pathways and a rostrocaudal enteric neural innervation gradient allows gastric slow waves to oscillate with a moderate range of frequencies and to propagate with a broad range of velocities, thus preventing decoupling observed in motility disorders. Overall, the findings provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach, offer directions for future experiments and theoretical work, and can potentially aid in the design of new interventional pharmacological and neuromodulation device treatments for addressing gastric motility disorders.Peristalsis, the coordinated contraction—relaxation of the muscles of the stomach is important for normal gastric motility and is impaired in motility disorders. Coordinated electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICC) and smooth muscle (SM) cells of the stomach wall as a slow-wave, underly peristalsis. Normally, the gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. Understanding of the integrative role of neurotransmission and intercellular coupling in the propagation of an entrained gastric slow-wave, important for understanding motility disorders, however, remains incomplete. Using a computational framework constituted of a novel gastric motility network (GMN) model we address the hypothesis that engaging biological oscillators  by constitutive gap junction coupling mechanisms and enteric neural innervation activated signals can confer a robust entrained gastric slow-wave. We demonstrate that while a decreasing enteric neural innervation gradient that modulates the intracellular IP The coordinated contraction and relaxation of the muscles of the stomach, known as peristalsis is important for normal gastric motility and primarily governed by electrical depolarizations that originate and propagate within a network of interconnected layers of interstitial cells of Cajal (ICCs) and smooth muscle cells of the stomach wall as a slow-wave. Under normal conditions, a gastric slow-wave oscillates with a single period and uniform rostrocaudal lag, exhibiting network entrainment. However, the understanding of intrinsic and extrinsic mechanisms that ensure propagation of a robust entrained slow-wave remains incomplete. Here, using a computational framework, we show that in conjunction with an enteric neural innervation gradient along the rostrocaudal ICC chain, and intercellular electrical coupling, the intercellular exchange of inositol trisphosphate between ICCs prevents decoupling by extending the longitudinal entrainment range along the stomach wall, even when variability in intercellular coupling exists. The findings from our study indicate ways that ensure the rostrocaudal spread of a robust gastric slow-wave and provide a mechanistic explanation for the emergence of decoupled slow waves associated with motility impairments of the stomach. Gastric peristalsis, the coordinated contraction and relaxation of the muscles of the stomach, is a critical phenomenon for food propulsion and waste product elimination  and is iPeristalsis is governed by diverse and overlapping mechanisms which normally ensure robust motility patterns. Peristalsis emerges from a mutually coupled chain of pacemaker cells called the interstitial cells of Cajal (ICCs). The ICCs can independently generate electrical activity known as pacemaker potentials, which drive rhythmic potentials in the circular and longitudinal smooth muscle (SM) cells embedded in the stomach wall ,11–13. TThe propagation is enabled in part by gap junction channels between the ICCs, between ICCs and SM cells, as well as between SM cells –17. The 2+) and inositol 1,4,5- trisphosphate (IP3) within their cytoplasm i). The bold arrows in the schematic show the inflows and outflows of the key model variables  [IP3−Ca2+ feedback in Ca]SS,i increases a non-selective cation channel current (NSCC) which closes the loop by impacting the membrane potential (the Ca2+−Vm feedback)  results in an increase in the [Ca]SS,i mimicking the endogenous release of Ca2+ from IP3-operated stores in the ER. In response to [Ca]SS,i increase, the Ca2+ uniporter on the mitochondrial membrane is gated open, and Ca2+ ions flow into the mitochondria down the steep electrochemical gradient (the term JUni in Methods represents this mechanism). This is thought to remove a larger number of Ca2+ ions from the submembrane space than had previously entered from the ER, causing a temporary drop in the subspace Ca2+ concentration [Ca]i due to opening of voltage-dependent Ca2+ channels is followed by activation of voltage- and Ca2+-dependent K+ currents to cause repolarization that restores Vm to hyperpolarized values. The above events repeat to cause the regenerative pacemaker potential activity.An initiating event for pacemaker activity  is assumed to increase the IP neurons  and subs neurons ,51. In ontration ,49,50. Tβ) that modulates IP3 production along the ICC chain .The overall framework allowed us to examine whether engaging adjacent ICCs via two distinct mechanisms would result in similar entrained slow-wave. One mechanism relies on the electrical conductivity of the gap junction coupling between ICCs which  studies ,19. The ever see ). In ourever see ,23,25; t2+]i results in the depolarization of ICC and a subsequent upstroke of ICC membrane potential. The abrupt change in [Ca2+]SS is due to the sharp rise of Ca2+ in the submembrane space because of a Ca2+ efflux from ER and the subsequent sharp drop because of Ca2+ movement into the mitochondria. Compared to the [Ca2+]SS change, the change in intracellular IP3 concentration is relatively gradual. Although each OU has its own intrinsic frequency of oscillation, OU intrinsic frequency is linearly dependent on the enteric neural innervation  mV, which is very close to the value reported in i), submembrane space Ca2+ ([Ca2+]SS), and intracellular IP3 concentration ([IP3]).The 4 key state variables for an ICC-SM oscillatory unit have been plotted for 2 representative cycles. The state variables are: membrane potential of an ICC (V(TIF)Click here for additional data file.S2 Fig(A) Intrinsic frequency of all 42 OUs have been plotted against the individual OU and its corresponding neural innervation. Intrinsic frequency of 42 OUs have been measured in cycles per minute (cpm) for the corresponding enteric neural stimulus received. (B) Intrinsic frequency of an oscillatory unit is linearly dependent on the enteric neural innervation, β. The best-fit line follows a linear equation of the form: y = Ax+B, where A = 3.563e5, B = −5.937; R2 = 0.9959.(TIF)Click here for additional data file.S3 Fig(A) The network. (B) Spatiotemporal map of membrane potential (B1), Relative Lags(B2), and spatiotemporal map of SM Cell Periods(B3) for the network when -1 (left panel diagrams) and 0.2 sec-1 (right panel diagrams). (C) The Total Lags for changes in -1 are shown in the legend. (D) For several networks, the mean Total Lag(D1) and the SM Cell Period(D2) of the last 7 cycles for each network with respect to its (TIF)Click here for additional data file.S4 Fig(A) ICC-SM cell electrical conductance does not have any effect on network entrainment evident from the approximately equal values of Total Lag measured for 4 different values of ICC-SM cell electrical conductances. (B) Membrane potential of 4 equidistant ICCs (top diagrams) and SM cells (bottom panel diagrams) in the 42-cell network when ICC-SM cell conductance is 0.5 ns (left) and 0 ns (right). (C) Spatiotemporal map of membrane potential of all 42 SM cells of the network, where ICC-SM cell conductance is 0.5 nS (left) and 0 nS (right). (D) Reduction of ICC to SM cell gap junction conductance reduces the amplitude of SM cell membrane potentials. For representation purposes, here the amplitudes  of membrane potentials of the first and last SM cells of the network are shown.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table. The asterisk represents the GICC−ICC value used in the default network in Mean ± standard deviation for the last 7 cycles in each simulation (DOCX)Click here for additional data file.S4 TableMean ± standard deviation for the last 7 cycles in each simulation.(DOCX)Click here for additional data file.S5 Table. The asterisk represents the PIP3 value used in the default network in Mean ± standard deviation for the last 7 cycles in each simulation (DOCX)Click here for additional data file.S6 TableMean ± standard deviation for the last 7 cycles in each simulation.(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Irreversible destruction of the hair follicle (HF) in primary cicatricial alopecia and its most common variant, frontal fibrosing alopecia (FFA), results from apoptosis and pathological epithelial-mesenchymal transition (EMT) of epithelial HF stem cells (eHFSCs), in conjunction with the collapse of bulge immune privilege (IP) and interferon-gamma-mediated chronic inflammation. The scaffolding protein caveolin-1 (Cav1) is a key component of specialized cell membrane microdomains (caveolae) that regulates multiple signaling events, and even though Cav1 is most prominently expressed in the bulge area of human scalp HFs, it has not been investigated in any cicatricial alopecia context. Interestingly, in mice, Cav1 is involved in the regulation of (1) key HF IP guardians , (2) IP collapse inducers/markers , and (3) EMT. Therefore, we hypothesize that Cav1 may be an unrecognized, important player in the pathobiology of cicatricial alopecias, and particularly, in FFA, which is currently considered as the most common type of primary lymphocytic scarring alopecia in the world. We envision that localized therapeutic inhibition of Cav1 in management of FFA , could inhibit and potentially reverse bulge IP collapse and pathological EMT. Moreover, manipulation of HF Cav1 expression/localization would not only be relevant for management of cicatricial alopecia, but FFA could also serve as a model disease for elucidating the role of Cav1 in other stem cell- and/or IP collapse-related pathologies. Primary lymphocytic cicatricial alopecias (PLCA) are characterized by progressive, permanent hair loss for which there is currently no cure ,2,3,4. PFrontal fibrosing alopecia (FFA) is now the most frequent form of PLCA , which ep < 0.001). Lastly, it has been suggested that beta-blockers indeed can cause (in a minority of patients) telogens effluvium due to premature catagen induction in anagen HFs and that beta blockers profoundly regulate HF cycling (in mice), which also prominently express beta2 adrenergic receptors in their stem cell zone (bulge) [There has also been a proposed association between beta-blockers and lichen planus, lichenoid drug reactions and LPP , while C (bulge) .Histologically, lesional HFs in FFA display a lichenoid inflammatory cell infiltrate and perifollicular fibrosis with eventual replacement of the entire HF with fibrous tissue (fibrotic tracts) . In contUnderstanding the mechanisms by which a relative IP niche in the bulge  is creatEpithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose polarity, cell-to-cell contact and acquire a mesenchymal phenotype. Interestingly, lesional FFA HFs show cells within the bulge epithelium that are abnormally positive for the EMT markers  , mesenchIn this context, the plasma membrane of eHFSCs, namely its caveolae and their principal structural component, caveolin-1 (Cav1) ,50, has We have previously shown that Cav1 localizes to the basal layer epidermal keratinocytes where it antagonizes keratinocyte migration and proliferation. Subsequently, spatiotemporal downregulation of Cav1 is required for appropriate cutaneous wound healing, and topical formulations which perturb Cav1 can accelerate wound closure using in vitro human skin organ cultures, human ex vivo wounds, as well as both murine and porcine in vivo wounds ,56. GiveImmunohistologically, the bulge area of the human HF displays high mRNA and protein levels of K15 in eHFSCs ,64, whicImportantly, numerous reports have demonstrated that Cav1 antagonizes guardians of HF-IP  ,71, and Furthermore, not only has expression of Cav1 been demonstrated to be upregulated during EMT ,87, but KO), we observed that Cav1KO mice exhibit an upregulation of IP guardian genes (CD200 and IL-10) and a downregulation of IP collapse and IFNγ-related genes   and a downregulation of EMT-related gene TWIST1  or Cav1 global knockout mice (Cav1 CXCL11) . Furthere TWIST1 . The phye TWIST1 , owing tSpecifically, we hypothesize that upregulation of Cav1 as exhibited in human FFA samples  inhibits®  an inducible Cav1 knockout mouse model under the control of the K15 promoter to specifically ablate Cav1 in mouse eHFSCs; (2) full-length human HF organ culture in which HF IP collapse and pathological EMT can be experimentally induced in healthy eHFSCs ,18 in threatment , statinsreatment  and siRNOne elementary requirement for validating the above hypothesis obviously is that Cav1 is expressed at all in the bulge region of human scalp HFs, i.e., in the epicenter of FFA pathobiology. Indeed, preliminary data we have just generated suggest that this not only is the case, but that Cav1 is very prominently expressed in the bulge epithelium, where it co-localizes with the key human eHFSC markers ,66, kera®, a selective agonist of olfactory receptor 2AT4, which is expressed also in the bulge, significantly prolongs human hair growth (anagen) by upregulating the intrafollicular production of IGF-1 [It would also be very good to know whether downregulating Cav1 in human scalp HFs is overall hair growth-inhibitory or, as our hypothesis predicts, hair growth-promoting. In addition, a recent study from Prasad et al. , on the of IGF-1 . PrelimiNext, the hypothesis can be probed by examining whether downregulation of Cav1 in the HF  have shown that methyl-β-cyclodextrin (MβCD) downregulates Cav1 expression by removing membrane cholesterol and preventing oligomerization of Cav1, the cell membrane ,40. Our The arguments delineated above render it likely that investigating the regulation/modification of Cav1 expression/localization in HFs will identify a powerful novel regulator of human eSC biology in general, and of human eHFSCs in particular. Moreover, the suggested research focus may identify in Cav1 a promising novel target for therapeutic intervention—not only in FFA management, but also for other forms of cicatricial alopecia , as well2+ increase [There is, however, a striking resemblance between topical Sandalore treatment and Cav1 depletion in skin including increased cAMP activity and subsequent Caincrease , activatincrease ,56,105. Gk-5-deficient mice  disrupts hair follicle development and cycling in these mice, which can be partially rescued by localized simvastatin treatment. Cakmak et al. [Since statins have been utilized extensively for a number of diseases (skin and others) over the past few decades, the ease at which they could be repurposed for treatment of FFA, and possibly other forms of scarring alopecia, makes them an attractive option. It should be noted, however, that sustained topical usage of statins may lead to aberrant sebaceous gland activity and sebum composition. Unfortunately, our current understanding regarding the role statins may have in sebaceous gland activity is limited and based on mouse models. For example, Zhang et al.  demonstrk et al.  demonstrk et al. ,108. SinProbing our hypothesis on the role of Cav1 in the development of FFA and other PLCAs and the importance of downregulating it to strengthen and/or restore IP in the bulge of human HFs  may not


Lower back pain and musculoskeletal injuries are serious concerns for workers subjected to physical workload and manual material handling tasks. Spine assistive exoskeletons are being developed to support the spine and distribute the spine load. This article presents a detailed up-to-date review on the back support exoskeletons by discussing their type (Active/Passive), structure (Rigid/Soft), power transmission methods, weight, maximum assistive force, battery technologies, tasks , kinematic compatibility and other important features. This article also assesses the back support exoskeletons in terms of their ability to reduce the physical load on the spine. By reviewing functional and structural characteristics, the goal is to increase communication and realization among ergonomics practitioners, developers, customers, and factory workers. The search resulted in reviewing 34 exoskeletons of which 16 were passive and 18 were active. In conclusion, back support exoskeletons have immense potential to significantly reduce the factors regarding work-related musculoskeletal injuries. However, various technical challenges and a lack of established safety standards limit the wide adaptation of exoskeletons in industry. Musculoskeletal disorder (MSD) is a type of chronic physical issue resulting from repeatedly lifting heavy objects by the labor at work. It is the cause of an inordinate number of missed workdays, workers' compensation claims, skyrocketing medical bills, lost productivity, and even early retirement. Annually over 40% of the laborers in the EU agonize lower back pain due to overexertion of manual handling tasks. Most of the personnel are subjected to physical workloads due to manual handling of material, awkward body postures, and repetitive movements causing musculoskeletal injuries . In the A few literature reviews have articulated the technical aspects of the back support exoskeletons. Looze et al. review provided an outline of assistive exoskeletons which were particularly designed for industrial applications and evaluate the prospective outcome of these assistive exoskeletons in terms of physical load reduction on the wearer . Toxiri This article presents a systematic review of current back-support devices focusing on their type (Active/Passive), structure (Rigid/Soft), actuation technology, weight, power, battery technologies, tasks , kinematic compatibility, and other important features. This article also assesses the potential effects of back-support devices on physical load reduction of the spine. Design choices for each of these features determine the user comfort, cost-effectiveness, complexity, and biomechanical effectiveness of the resulting exoskeletons. By reviewing the corresponding factors, the aim is to develop a better understanding and enhance communication between developers, ergonomics experts, and factory laborers.This article presents a systematic review of the back-support exoskeleton by following PRISMA guidelines. Records are compiled by searching through several databases such as IEEE digital library, Scopus, PubMed, ASME digital library, Medline, and additional records from other sources. Naming convention in these devices is not consistent and they are mostly named based on their developer or associated research centers, regardless of their purpose and technology involved. Hence to cover maximum devices, several keywords are used which are: assistive exoskeletons, industrial exoskeletons, rehabilitation robotics, assistive technologies, wearable robotics, and back-support devices.References that report the planning phase of devices and haven’t yet taken any physical form were excluded from the review. This review is compiled of references from conferences, journals, and other commercially developed devices.Various data variables were considered for data extraction such as actuation type, structure, force transmission methods, kinematic compatibility, tasks, power, maximum assistive force, and physical load reduction of the devices. This study aims to address following research questions (RQ):RQ1: What are the functional and structural features of the back support exoskeletons?RQ2: What sort of force transmission methods are available for back support devices?RQ3: How good back support exoskeletons are in terms of physical load reduction?RQ4: What role does kinematic compatibility play in exoskeleton design?RQ5: What are the major challenges faced by exoskeletons regarding wide industrial adaptation?Initial searches result in 419 records in total. After the elimination of the duplicate references, the records are further reduced to 329. The records were then further shortlisted by screening the titles and abstracts. This results in 77 records, which were further reduced by eliminating reference due to reasons stated in Back-support exoskeletons are designed to offload the spine by contributing a portion of the required torque for the completion of a physical task. These devices aim to assist back extension and, in some situations, hip extension. The forces and torques could be exerted in different ways to the subject. The one key difference between current systems is the direction of the forces, which are either perpendicular or parallel to the spinal column. A force parallel to the spinal column, in addition to assisting lower back extension, also contributes to internal spine loading which is an unwanted compression on the vertebral column. However, a perpendicular force does not contribute to spinal loading . Figure In the development of back support exoskeletons, kinesiological considerations imply that the lumbosacral (L5/S1) region of the spine encounters massive mechanical loading and peak compression forces in a variety of activities. . CompresAs the properties of these assistive exoskeletons vary substantially with the way they are designed. They can be classified based on their actuation (active/passive), structure (rigid/soft), and the degree that the device fits or goes with the person’s anthropometry (kinematic compatibility). The selection of actuation mechanisms may define their use in different tasks. Passive exoskeletons seem more appropriate for tasks involving comparatively little assistance and less dynamic activities. On the contrary, heftier and vigorous tasks will vindicate the use of more complex active exoskeletons. The majority of back-assist devices are designed using rigid modular structures, which are hefty and bulky but produce more suitable patterns of torques and forces. A few soft robotic suits have been designed in recent years, they offer better user comfort and can be worn underneath the dress or integrated with working wear. The adoption of any assistive device will eventually depend on various factors, comprising of user acceptance, costs, and benefits associated with a specific application . Table 1Based on actuation, back support exoskeletons can be catogorized as active or passive. Active assistive exoskeletons are driven by electric motors, pneumatic muscles, or hydraulic actuators, whereas the passive assistive exoskeletons often comprise cheaper mechanisms such as metal or gas springs, elastic elements, etc. These actuation or power transmission mechanisms define the structure (rigid/soft) of the exoskeletons.Passive devices such as Laevo , BackX , WMRD W, BNDR U; 2013a, Passive wearable devices are designed to provide maximum strength and offload the spine. Another design factor to consider in such devices is bulkiness. The devices may support lifting heavier items but with a bulky device, the user may experience discomfort. Devices such as Spexor and BackX weighing from 3.5 to 6.7 kg are heavier compared to soft suit-like devices such as Smart Suit Lite , WAD He, B.A GarActive devices such as RoboMate , HAL LumSafety has always been one of the major concerns in human-robot interaction and it is directly related to actuation technology. Therefore, compliant actuators such as series elastic actuators (SEAs) and parallel elastic actuators (PEAs) are widely adopted because they offer a variety of advantages over conventional rigid actuators. ExMS-1  was deveReducing the metabolic cost and weight of the device is one of the key challenges faced by the exoskeleton developers. An actuation mechanism that consumes less power and has a compact lightweight design would be ideal for wearable applications. One such actuation system is the twisted string actuator (TSA). TSA converts the rotation of the motor into linear motion with help of twisted strings coupled to its shaft. One can generate a higher pulling force with low-power motors. This combination results in mechanically simple, low power, and lightweight actuators . A twistCurrent active spinal assistive exoskeletons use motors or pneumatic actuators. These actuators are bulky and not very human interactive. Using variable stiffness mechanisms to develop the exoskeletons has huge potential. Layer jamming-based structures are emerging with a new set of possibilities among the variable stiffness mechanisms . Layer jRegarding force transmission, the development of passive devices is relatively less costly, lightweight, and simpler to implement in comparison to active devices. Active devices on the other hand are more versatile and have the potential to be employed in a variety of scenarios.A key element, that differentiates assistive exoskeletons, is the extent of alignment of the device’s kinematic structure with the user body. Misaligned joints can reduce comfort and generate undesirable torques and forces of up to 1.5 Nm and 230 N, respectively . Since tA certain level of misalignment is permissible and can be compensated by bringing more degrees of freedom to an exoskeleton. Moreover, appropriate compensation of misalignment precludes relative motion between the exoskeleton and the body, thus obliquely enhances comfort . IntroduOne must also consider the kinematic compatibility in the transverse and coronal planes while designing the exoskeleton for back support. The laevo exoskeleton features a chest pad with rotational elements to achieve somewhat differential transmission. RoboMate exoskeleton relatively incorporates the misalignment compensation in a more elaborated way, comprising of one ball and two hinge joints. A similar compensation mechanism is employed for the trunk . The plaIndustrial exoskeletons used for the assistance of lifting and bending tasks generally include a powered flexion-extension hip joint, which moves only in the sagittal plane. They are unable to provide assistance in the frontal plane. To overcome this, SuitX developed two different models (“S” and “AC”) of the BackX exoskeleton for lumbar support . BackX mSoft robotic suits have been developed and optimized for years to enhance comfort and kinematic compatibility. These devices are lightweight and mostly passive. The Personal Lift Augmentation Device (PLAD) was one of the earliest soft back-support exoskeletons, which uses tension in elastic bands to transmit the forces . AlthougPresence of rigid structures in exoskeletons alter the lifting technique due to misalignment between exoskeleton and human joints resulting in altered joint kinematics . Unlike The studies reporting the effects of back support exoskeletons on spine loading rely on a set of outcome measures. A metric used frequently is the rate of muscular activity reduction. Which is the outcome from wearing the device, measured by the myoelectric activity at the targeted muscles. Passive devices are designed to offload the lumbosacral (L5/S1) joint and seem to be very effective for both static holding and dynamic lifting tasks. The type of actuator and structure play a significant role in the reduction of muscle activities. The passive back support devices can be divided based on their type of assistance and rigidity. This section is categorized based on the type of assistive technology used in passive devices and their impacts on muscle activity reduction.The impact of spring-actuated devices on muscle activity reductions is discussed in this section. Torque is generated between the torso and the thigh linkages by springs. This torque exerts a force on the user, compensating for the torque created by the user’s torso weight. The effect of the static holding task was investigated for the device Laevo which uses gas springs for assistance. The studies showed positive effects on lowering muscle activity during static trunk activity by 35–38%, also reduced discomfort in hip extensor activity. However, the discomfort was reported in the chest region . BackX aBNDR uses torsional springs to reduce compression and shear forces for stooped posture. Reductions of 13.5 and 12.1% were observed in compression and shear forces at L5/S1. This results in the reduction of load at spine tissues and ultimately reduces the risk of injury ; 2013a. All the devices discussed in this section are spring actuated with rigid structures except Passive Spine Exoskeleton which has a semi-rigid structure. Rigid structures are reported to raise issues such as an increase in leg muscle activity, increase in muscle deconditioning, and discomfort. For a device like the Laevo, an overextended position of knees during the holding task was observed which can lead to health risks if the exoskeleton is used for longer periods.The spring-actuated exoskeletons can only assist in flexion/extension movements while limiting motion in other planes. To increase the range of motion in other planes, a concept for actuation consisting of multiple continuous carbon fiber beams was introduced. Spexor used flexible beams running parallel to the spine to allow a higher range of motion. It was able to reduce the torque requirements with compensation of misalignments for both hip and lumbosacral joints. Compared to other passive devices, only a moderate reduction in the back muscle forces (18–25%) was observed, while abdominal muscle forces increased. This may be due to the dynamic activity during the trials rather than the static .VT-Lowe’s utilizes carbon fiber-based flexible beams running in parallel to the spine up to the back of the thigh. It significantly reduced activity in the spinal muscles (29.3%) during symmetric and asymmetric lifting .. HoweveThe majority of passive devices utilize elastic components as an external power generator positioned next to the erector spinae and thigh muscles. These elastic components transmit a portion of the spine’s forces and moments to the shoulders, pelvic girdle, and knees. As the upper body weight is decreased during lifting tasks, the elastic elements store energy, which is then released during the upward phase, decreasing the energy demand on back muscles. PLAD is a soft wearable device that uses elastic bands to assist the spine. A reduction in compression and shear forces by 14–29% was observed during various tasks. However, 40% of the individuals participating in the trial reported discomfort while wearing the device .APEX exoskeleton incorporates a proprietary auto clutch with elastic bands to activate or deactivate the assistance. A significant reduction was observed in lumbar muscle activity and fatigue rate by 43% and 19–85% respectively . WAD, anHappy Back, a semi-rigid passive suit utilizes bungee cords to assist the motion. It offers a reduction in the erector spinae muscle activity by 23% . Unlike Reduced back muscles activity may be accompanied by increased activity of other muscles, depending on the lifting method. An increase in Tibialis Anterior Activity (TAA) was reported for BNDR. The fact that, in both, static holding and dynamic lifting activities, external forces exerted by the device must be countered to maintain balance might explain the increase in leg muscle activation. In prolonged lifting and lowering work, increased leg muscle activity could significantly increase the metabolic demands and oxygen uptake. However, for VT-Lowe’s, the reduction of ∼7.9% metabolic demands and ∼8.7% oxygen uptake was reported.Active wearable devices are potentially more versatile as compared to passive devices. Their capabilities are dependent on the activation type and the utilized strategy to support the lumbosacral joint. Most of the devices are powered by electric motors and a few using pneumatic actuators.The majority of the active back support devices are rigid and use electric motors as a mode of actuation. Several studies have been carried out focusing on the reduction of muscular activities for static and dynamic activities of these devices. HAL, a hybrid assistive limb was developed to support muscles in the lumbar region during repetitive lifting tasks. A significant amount of reduction was observed in the myoelectric activity of the thoracic and lumbar erector muscles by 19.3 and 14% respectively .Hyundai H-WEX is another rigid exoskeleton that uses electric motors to assist the spine. The reduction in muscle activity of Hyundai H-WEX was observed to be 23.5, 18.6, and 30% for erector spine, gastrocnemius medialis, and biceps femoris . A similThe easy-to-wear SIAT waist exoskeleton is capable of reducing muscular activities around lumbar and thoracic spinal muscles by 33.9, 34, and 4.1% for TES, LES, and LD muscles respectively . During The Exo Muscle Suit, an active pressure-driven rigid active device uses artificial McKibben pneumatic artificial muscles (PAMs). The suit is supporting mainly the erector spine muscle group and is reported to reduce muscular activity by 42.6% . The waiAnother soft active device (WSAD) uses tension bands driven by servo motors was developed mainly for stooped postures. It reduced muscle activity of TES, LES, LD, and RA by 42, 47, 28, and 9%, respectively .Maximum reductions of muscle activity for active back-support devices were observed for Soft Power Suit. The device is equipped with twisted string actuators, making it lightweight and capable of generating high forces in a linear direction. The muscle activity reduction measured for the erector spine, biceps femoris, gastrocnemius medialis, and semitendinosus was 69.2, 35, 63.6, and 48.25% respectively .To summarize, both passive and active exoskeletons, help in muscle activity reduction at the lumbosacral (L5/S1) area, making any physical task simpler to accomplish. In this analysis, a passive exoskeleton called Paexo was shown to have the highest reduction in muscle activity. This might be because the experiment only investigated one posture. Further experiments including dynamic tasks would give a more comprehensive perspective of the muscle activity decrease for PAEXO. An active device (Soft Power Suit) on the other hand, allows for considerable decreases in muscular activity while executing a dynamic task. In terms of their impact on physical load, these studies suggest that they all can reduce muscular activity in the lower back for varying tasks . Another potential challenge many active devices face is the bulkiness of the device itself, mainly due to the weight of actuators and batteries. However, there are devices such as Soft Power suit, Waist assist suit, Spine inspired continuum soft Exo, WSAD, Japet (Atlas), and HAL which weigh under 3 kg. A device such as the Lower back robotic exoskeleton that weighs approximately 11.2 Kg excluding batteries may prove to be discomforting in the long run.Passive exoskeletons are generally user-friendly, cost-effective, lightweight (1.5–3 Kg), and provide the intended range of motion. These aspects have caused passive exoskeletons to gain immense interest among ergonomics, human factors, robotics, and biomechanics researchers as well as captains of industry as a potential solution to prevent musculoskeletal back injuries. Few issues have come into the light regarding the potentially negative effects related to higher leg muscle activity, muscle deconditioning, and high levels of discomfort. On the contrary, new active exoskeletons have emerged in the market (Hyundai H-WEX5 and ATOUN Model A4), as industries have invested significantly in this area. Even though active exoskeletons have more design complexities, they have biomechanical advantages compared to passive devices because of their intrinsic versatility.Several factors have been identified in previous studies to be responsible for lesser adaptability of the back support exoskeletons: kinematic incompatibility , loss ofThe real effect on potentially preventing back injury occurrence, yet to be evaluated, as till today substantial technical challenges and inadequate safety standards hinder the large-scale implementation in workplaces.This article comprises a systematic review of existing back-support devices in three distinct functional and structural features which are 1) actuation and structure, 2) kinematic compatibility, and 3) Reduction of muscular activities. Design choices for each of these features govern the comfort, biomechanical effectiveness, complexity, and cost-effectiveness of the resulting exoskeletons.In terms of actuation, we hypothesize that passive exoskeletons are less complex, less expensive, and more lightweight than active exoskeletons. Active devices, on the other hand, offer a greater potential for versatility and hence a larger range of applications. Open technological challenges for active exoskeletons include optimization of the control to utilize their versatility to broaden their potential impact . Soft roBack support exoskeletons have the potential to significantly minimize the underlying causes that lead to work-related musculoskeletal pathologies. However, the real impact on possibly lowering injury prevalence has yet to be shown, as large-scale adoption in workplaces has been hampered by major technological hurdles and a lack of specified safety requirements.


When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K+ channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca2+ transients, we, in mature trigger hairs firing fast Ca2+ signals and APs, found OSCA1.7 and GLR3.6 type Ca2+ channels and ACA2/10 Ca2+ pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca2+ and electrical event. Given that anesthetics affect K+ channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca2+ and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca2+ activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca While APs in Arabidopsis and most other plants last > 1 min and cannot be evoked repeatedly2, the flytrap equivalent takes just about a second and can be fired a maximal frequency of 1 Hz3. Compared to nerves with the information encoded by the frequency (number of APs per time), the flytrap system is more remote. It, however, allows Dionaea count to five APs4. When a potential prey visiting the trap, attracted by color and odor, touches one of the six trigger hairs, an AP gets fired. The trigger hair is a mechano-sensitive organ that gets excited by share stress of > 3° bending of 29 µN force5. Two AP make the trap close and imprisons the animal prey. Three and more APs trigger touch hormone jasmonate (JA) signaling and JA production. A count of 5 is required to produce digestive enzymes and transporters that are specialist to take in the animal derived nutrients8.Within the plant kingdom the action potential (AP) of the carnivorous Venus flytrap is most similar to the all or nothing AP in our nerve cells10). MSL10 type channels in Arabidopsis and Dionaea operate as anion channels that upon activation depolarize cells11, while OSCAs rather Ca2+14. To trigger excitation in the flytrap cells have to be depolarized by 20–40 mV from the resting state15. Thus, together MSL10 and OSCA(s) likely give rise to depolarization and Ca2+ influx for activation of anion channels. Anion channels in plants are the master switches in plant electrical excitation. The opening of anion channels and thus membrane electrical excitation is ultimately linked to the amplitude and kinetics of the given stimulus induced Ca2+ transient. Initial notice of a calcium-voltage-change association was made in the context of the wound/foraging response in the model plant Arabidopsis. Upon wounding the signaling molecule glutamate gets released to the extracellular space16, faced by ligand-binding site of GLRs18. The Arabidopsis genome harbors 20 GLRs. Edgar Spalding lab in 2006 was first to demonstrate that glutamate evokes a Ca2+ signal as well as membrane depolarization which is suppressed when GLR3.3 is mutated19. The Ted Farmer and Simon Gilroy labs have unequivocally shown that the electrical- and Ca2+ signals, moving from the injured side in the local leaf to those wired to it via the vasculature, require the presence of the two GLR3.3 and 3.6 glutamate receptors16.Hapto-electric energy conversion takes place in the indentation zone of the trigger hair podium. In this zone mechano-sensitive channels of the DmMSL10 and a DmOSCA1 type are expressedDionaea traps insensitive to touch20 and even Mimosa’s movement gets inhibited by ether21. Andrej Pavlovič and colleagues very recently have documented that an ether atmosphere suppresses AP firing and trap closing in Dionaea22, while direct application of liquid ether on the trap evokes APs leading to trap closure23. Cation channels responsible for the excitability of nerve cells are targets of anesthetics such as ether25. Key for anesthetics susceptibility are channel sites interacting with membrane lipids26.Already in 1875 it was described that ether turns 2+ and AP stimulated trigger hairs excite capture organ. The fact that DmGLR3.6 expression was associated with flytrap excitability and the anesthetics suppressed glutamate-induced signals too, pointed to GLRs as likely ether targets.Here we asked the flytrap ion channel target of ether. We found ether to suppress CaDionaea trigger hairs can operate as sender and receiver of touch induced action potentials and Ca2+ waves27. How is this possible? The flytrap excitable cells rest around − 140 to − 120 mV. Whenever the membrane potential is depolarized below − 100 mV an action potential is evoked.Our recent studies have shown that the For technical reasons one cannot measure APs in the very trigger hair and mechanical stimulate it at the same time. However, when voltage-recording microelectrodes were inserted in to the oblong mechano-sensitive cells of the podium of a receiver trigger hair, we could receive the AP induced by touch stimulation of one the other two mechanosensory organs operating as senders Fig. .2+ signal can be recorded in the same touch stimulated organ. Two types of Ca2+ signals were picked up: (i) upon sub-threshold trigger hair bending the podium cells emitting GCaMP6f fluorescence first were those of indentation zone prone to sheering stress, while (ii) above-threshold stimulations lighted up the entire podium that gave rise to selfsustained Ca2+ signal that left the trigger hair and propagated along the trap surface . The calcium fluorescence faded away in the indentation zone first, a finding in agreement with the notion that cells that were activated initially are subject of bleach first. This Ca2+-signal in the sender trigger hair and two receivers moved with a velocity of 2.66 ± 0.18 mm/s  from the podium toward the hair tip.In contrast to the AP the Caace Fig. . In respace Fig. . Followi2+ signal, one lobe was cut from a Dionaea trap mounted on a microscope stage. The sender trigger hair was stimulated every 1 min with ether fumigation initiated after the third AP  involved in mechano-transduction and shaping the Ca2+ transient in the trigger hair podium do not present ether targets.To investigate the ether effect on the Ca AP Fig. . Already2+ wave coming back. Initially it did not travel the full trap surface, but the area right hand side of the capture organ displayed in the video : (i) an initial fast depolarization phase to − 60 mV which was followed by (ii) a slower phase reaching the peak depolarization voltage of − 20 mV. The depolarization phase is composed of (iii) an initial fast component during which the membrane voltage repolarizes to − 60 mV and (iv) an initial lower component restoring the pre-stimulation voltage of − 120 mV that merges with an even slower, (v) hyperpolarization phase that peaked at − 130 mV. From this hyperpolarization overshoot the membrane potential in (vi) final, a yet slow recovery phase returned to its pre-stimulus resting level again.To study how ether affects the AP, we have placed a + ATPase30. The depolarization phase of the Dionaea AP results from Ca2+ influx and Ca2+ activation of the anion channel33. In presence of the general Ca2+ channel inhibitor La3+ touch induction both action potential34 and Ca2+ wave is suppressed27. This indicates that both signals are interconnected and that the Ca2+ channel blocker La3+ as well as ether anesthetics is suppressing the transition of Ca2+ signal and AP from the trigger hair podium into the trap. Anesthesia, however, does not seem to target podium located mechano-sensitive Ca2+ channels and Ca2+ ATPases (addressed below).When exposed to an ether atmosphere the resting potential did not change much but 5 min after anesthesia onset the peak depolarization dropped to − 40 mV and the overshoot hyperpolarization vanished completely. The overshoot results from a depolarization amplitude dependent transient hyperactivation of the AHA type H10. Before trap had not opened during development it is regarded unmature and classified stage V  genes are expressed in the trigger hair10, (ii) expression is induced when trigger hairs maturate, developing trigger hair-only mechano-sensitivity and (iii) genes should predominately express in the podium.To spot the potential ether target, we reinvestigated and extended the RNA species that have been associated with CaDionaea trigger hair10, with some being specific to the trigger hair such as MSL10 and the Shaker K+ channels KDM1 and DmSKOR. Moreover, the glutamate receptors DmGLR3.4 and DmGLR3.6 as well as the Ca2+-ATPase DmACA2 were characterized as trigger hair specific as well. Apart from DmGLR3.4 all of these transporters we found predominantly expressed in the podium fraction of mature trigger hair  was described as trigger hair specific. However, the expression of this gene is rather low. We therefore re-analyzed our trigger hair transcriptome and identified another OSCA1.7 type mechanosensitive Ca2+-channel in the trigger hair which we named Dm (Dm_00005287-RA) based on phylogenetic analyses. DmOSCA1.7 is expressed in the trigger hair and the mature trap and induced in both tissues during trap maturation and gain of electrical excitability  and Ca2+ wave does not travel out of the locally wounded leaf. Furthermore, the Simon Gilroy lab has shown that upon wounding glutamate is released from injured cells16. When glutamate was applied to pre-wounded Arabidopsis leaves an electrical signal and a calcium wave was elicited19.In addition to DmOSCA1.7 we found DmGLR3.6 that represents homolog to Arabidopsis glutamate receptor channels GLR3.1/3.3 involved in long-distance mechanical stress electrical and Ca5) of GCaMP6f expressing Dionaea and monitored the calcium-electrical response. Following injury with a macroscopic pipette tip a rapid electrical- and calcium-signal was evoked that was spreading from the compressed trap site distally. In contrast to the stimulation of the trigger hairs (Supplementary Movie 2+ activate in the vasculature (c.f.16).Given glutamate release and GLRs are associated with wounding, we with an Eppendorf pipette tip pinched selected sites on traps -related a self-propagating Ca2+ wave, it springs up in the indentation zone, ingresses the podium, and spreads all over the trap. We have documented that the podium differentially expresses DmOSCA1.7 a homolog of the mechanosensitive Ca2+ channel AtOSCA137. In the working model . Upon mechanical activation of DmMSL10 anions are released and pre-depolarize the membrane. This voltage and OSCA/CICR Ca2+ above threshold activation initiated the depolarizing phase of the AP. The Shaker type voltage (depolarization) dependent GORK/SKOR type K+ channel is preferentially expressed in the podium and engaged with the repolarization of the AP. For the membrane potential to recover the resting state, the QUAC1 type anion channel the cytoplasmic Ca2+ level must drop. This task is accomplished by the two podium differentially expressed plasma membrane Ca2+ ATPase DmACA2 and 10 the ER Ca2+ pump DmECA1.We have shown that a threshold in the trigger hair displacement amplitude exists, that when passed gives rise to an APave Fig. A. Below 2+ transients, excluding an OSCA as target of inhibition. Ether anesthetics and the Ca2+ channel blocker La3+34, however, prevents the touch- and glutamate induced Ca2+ electric signal to exit the podium. A similar lack of mechanical and glutamate dependent initiation of long-distance calcium electrical signal is known from an Arabidopsis mutant that lost glr3.1/3.3. These two AtGLRs were found to express in the vasculature16.The primary site of action of general anesthetics is the nervous system, where anesthetics like ether inhibit neuronal transmission. Anesthetics directly target a subset of plasma membrane cation channels. Our studies have shown that ether does not impair touch induced CaDionaea genome encodes 22 GLRs and DmGLR3.6 is differentially expressed in the trigger hair podium. Upon glutamate application the vasculature emitted a particularly strong GCAMP6f. Ca2+ fluorescence emission but not in the presence of ether. It is thus tempting to speculate that in Dionaea DmGLR3.6 in the trigger hair podium and those expressed outside the mechano-sensor represent ether targets. This working hypothesis in future studies can be tested for DmGLR3.6 functionally complementation and anesthesia of the Arabidopsis glr3.1/3.3 mutant.The 2+]cyt increase a sample rate of 25 ms was chosen. Hairs and traps were background subtracted and the mean velocity was calculated over known distances. For quantitative analysis of areas with increased [Ca2+]cyt after stimulation two ROIs (regions of interest) with the same pixel numbers were defined in the indicated tissues using ImageJ. Data points were fitted using Igor Pro 8.Stereoscopic images and videos were taken using a Leica M165 FC fluorescence stereo microscope, a Leica EL6000 lightsource, the Leica filter set ET GFP LP , and a cooled CCD camera . For GCaMP6f. calcium imaging, an excitation wavelength of 475 nm and an exposure time of 50 ms at 2× binning was used. Fluorescence videos were taken as a sequence of uncompressed tiff files in a range where the fluorescence intensities of samples and brightness values of captured images were linear. Ether was applied by adding 2 ml ether to a glass container next to the sample in a gas-tight setup creating an ether atmosphere of ~ 15%. 10× time laps for Movies Dionaea muscipula GCaMP6f plants were provided from Mitsuyasu Hasebe lab27 and grown in plastic pots at 22 °C in a 16:8 h light:dark photoperiod. For microscopic experiments traps were bisected and the half which was still connected to the plant was fixed to a microscope slide the day before measurements.For the qPCR expression measurements of genes of interest in wild-type plants, the trigger hair was dissected in two parts: the basal part and the tip. Entire/un-dissected trigger hairs were also collected as a control. For the whole trigger hairs qPCR expression analysis 300–600 trigger hairs were needed for one replicate, while for the tip and the base parts between 880 and 940 trigger hairs were needed to extract enough RNA for one replicate. Three replicates were used in total n = 3.4-Ac (5 mM in EDTA), 60% Isopropanol , and water up to a final volume of 100 μl. The samples were washed using 70% Et-OH, centrifuged at 4 °C for 20 min, and the resulting pellet was dried at 37 °C and resuspended in water .For qPCR analyses, RNA was isolated from each sample using the NucleoSpin Plant RNA extraction kit  according to the manufacturer’s instructions and in combination with Fruit-mate for RNA Purification solution . Briefly, 100 mg of powdered plant material was thoroughly mixed with 350 μl of Fruit-mate solution for 1 min. Following 10 min of centrifugation  at 4 °C, the supernatant was mixed with the lysis buffer provided by the kit (RAP), together with 3.5 μl TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 0.5 M, pH 7, Sigma-Aldrich). Apart from this, the kit manufacturer’s instructions were followed except for the DNA digestion, which was performed in a separate step after the RNA isolation. For 1 μg of RNA, 3 μl DNase Buffer , 0.5 μl DNase inhibitor , and 1 μl DNase I , Thermo Fisher Scientific) were mixed in a final volume of 30 μl and incubated at 37 °C for 30 min. Next, the DNA-free RNA was precipitated in isopropanol overnight at − 20 °C, together with 1% glycogen (RNA Grade (20 mg/ml), Thermo Fisher Scientific), 10% NHD. muscipula genes of interest was performed by real-time PCR. D. muscipula transcripts were normalized to 10,000 molecules of DmACT1.The RNA was transcribed into cDNA using the M-MLV Reverse Transcriptase . qPCR was performed using a Realplex Mastercycler system  and ABsolute QPCR SYBR green capillary mix (Thermo Fisher Scientific). Quantification of the actin transcript DmACT1  and transcripts for Before the dry-impalement experiments, a trap was cut in half and the half that was still connected to the plant was fixed in a Petri dish on a chlorinated silver wire as a reference electrode with double-sided adhesive tape. After a 30-min recovery, the trap was inserted with a sharp impalement electrode. For impalements, microelectrodes from borosilicate glass capillaries with filament (Hilgenberg) were pulled on a horizontal laser puller  and filled with 300 mM KCl and connected via an Ag/AgCl half-cell to a headstage . An IPA-2 amplifier (Applicable Electronics) was used, and the cells were impaled by an electronic micromanipulator .–1 mol). The ether was placed in a glass vial next to the trap. The trap in Fig. To anaesthetise the traps, 1 ml of ether was completely evaporated inside a closed container reaching a concentration of ~ 15%. The approximate volume of diethyl ether was calculated using ideal gas constant (standard state of gas as 22.4 ll-glutamate was pipetted either onto the trap surface or onto the cut edge.To test the glutamate effect on the trap, 20 µl of 5 mM sodium For surface potential measurements in Fig. Supplementary Information.Supplementary Movie 1.Supplementary Movie 2.Supplementary Movie 3.Supplementary Movie 4.Supplementary Movie 5.Supplementary Movie 6.Supplementary Movie 7.Supplementary Movie 8.Supplementary Movie 9.Supplementary Movie 10.


This study aims to determine the influence of targeting araC-resistant acute myeloid leukemia by dual inhibition cyclin-dependent protein kinase (CDK9) and B-cell lymphoma-2 （Bcl-2).  The c-Myc inhibitor 10058-F4 and the CDK9 inhibitor AZD4573 were used to determine the cell cycle arrest and apoptosis.  10058-F4 reduces c-Myc protein levels and suppresses HepG2 cell proliferation, possibly by upregulating cyclin-dependent kinase (CDK) inhibitors, p21WAF1, and reducing intracellular alpha-fetal protein (AFP) levels.  The combination of AZD4573 and 10058-F4 has a synergistic anti-araC-resistant AML activity, providing a solid database for the aforementioned scientific hypothesis. Hematopoietic stem cells can self-renew and differentiate, and their control regulates their proliferation speed , 2. WhenThe Bcl-2 protein family comprises antiapoptotic proteins  and proapoptotic proteins , and the two classes of protein ratios determine cell survival or cell death , 14. TheMeanwhile, the apoptosis was induced by 10,058-F4 performing mitochondrial pathway activation, and downregulation of Bcl-2, upregulation of Bax, cytochrome C release within the cytosol, and cleavage of caspase 3, 7, and 9 were observed. In addition, bone marrow cell differentiation is also induced by10058-F4, perhaps by activating multiple transcription factors. Similarly, 10058-F4 acts on primary AML cells to cause apoptosis and differentiation. 10058-F4 reduces c-Myc protein levels and suppresses HepG2 cell proliferation, possibly by upregulating cyclin-dependent kinase (CDK) inhibitors, p21WAF1, and reducing intracellular-alpha-fetal protein (AFP) levels .L T 3 mutation-positive AML activity with ABT-199 [Moreover, Bcl-2 was also overexpressed in human AML stem cells, and inhibition of Bcl-2 selectively eradicated quiescent AML stem cells by inhibiting mitochondrial oxidative phosphorylation –25. The  ABT-199 . Still, CDK is a cyclin-dependent kinase that functions to activate the phosphorylation of downstream cell cycle-associated proteins while regulating its activity itself, influenced by phosphorylation and nonphosphorylation . Its actAZD4573 is a potent CDK9 inhibitor with high selectivity.  Scheme 1In cells, the short-term treatment of AZD4573 can decrease intracellular pSer2-RNAPII (concentration and time correlation), with the activation of caspase 3 and apoptosis in most hematologic blood cancer cell lines. In various human blood cancer cells, AZD4573 induced rapid caspase activation (6 h) and lost survival viability (24 h), with little effect on solid tumors. Cyclin-dependent kinases (CDK) are a wide range of key proteins that perform cellular activities and function, including cell division and transcription. Dysregulation is identified as an important tumorigenesis driver. Additionally, the role of CDK in cancer and its involvement in the transcriptional process has been demonstrated recently. In most cancers, CDKs openly used as vital transcriptional regulators  for consistent production of short-lived gene products that support cell survival . TherefoProtein kinases are an important name in the CDK9 inhibitors list. These are the large family of enzymes and are responsible for the regulation of most eukaryotic cellular functioning and regulate cell signaling pathways by protein phosphorylation. This process is responsible for enzymes activation or inhibition, increasing protein-protein interactions, alteration of cellular localization, or generation of protein recruitment sites. Regulation of protein kinases itself is generated by various transcriptional and posttranslational modifications. The overall outcome of the whole process is the regulation of cellular functioning, including proliferation, apoptosis, and differentiation. The encoding of the human genome includes more than 500 genes of protein kinase. Because of its key function, dysregulation of protein kinase due to gene mutations or lack of negative regulators is associated with many pathological diseases . A protein-dependent kinase (CDK) is a serine (Ser)/threonine (Thr) protein kinase activated by the regulatory cyclin. The encoding of 20 CDK (numbers 1–20) of the human genome belongs to the CDK and CDK-like clades of the CMGC. CMGC is a subfamily of human cell kinases, such as cyclin-dependent, glycogen synthase, mitogen-activated, and CDC-like kinases.G) rich loop structures of CDK. Due to the evolutionary connection and key functional roles, CDKs can be categorized into two major groups: CDKs that are responsible for the cell cycle regulation, including CDKs 1–7 and 14–18, and CDKs for transcription regulation, including CDKs 7–13 and 18–20. Due to these central regulatory activities, dysregulation of CDK is closely linked to human cancers or malignancies. This is especially true of CDK9, where CDK9 is a key transcriptional regulator in which cancer cells openly utilize to continuously produce short-lived proteins that sustain survival. During transcription elongation, a component called P-TEFb is a major target of DRB. A protein obtained from the human immunodeficiency virus 1 (HIV1), called the transcriptional transactivator (TAT), performs a vital role in HIV1 viral transcription by creating a complex containing P-TEF.CDK and its cyclin partners perform their particular roles in many cellular functioning, including cell division and cell transcription, responding to intracellular and extracellular cell signaling. The structure of CDK protein is a double-leaf, and the active site is sandwiched between the (amino) N-terminal lobe, particularly comprising sheet B and the (carboxyl) C-terminal lobe of the helix. The CDK regulation is initiated by cyclin subunits binding, which is itself regulated by formation and degradation, and the phosphorylation of the conserved residues in the T-and glycine  is a highly malignant hematological disease with abnormal proliferation, blocked differentiation, and high cell heterogeneity. Routine treatment of AML is divided into two stages. The first stage is the induced remission phase, and the primary aim is to reduce the number of AML main cells to achieve complete remission, where AML cells are not detectable in both the bone marrow and peripheral blood, and healthy blood cells return to normal levels. Commonly used drugs are cytarabine  and anthracyclines such as daunorubicin , idarubicin, and mitoxantrone. The conventional chemotherapy regimen was a 7-day continuous intravenous infusion of standard-dose cytarabine (100–200 mg/m2/d) plus daunorubicin continuous intravenous infusion (60–90 mg/m 2/d), also known as “7 + 3” therapy.The second stage of AML treatment is the postremission treatment. A small number of undetectable AML cells will still exist in vivo after complete remission. Without further treatment, AML will relapse within weeks to months. There are two strategies after remission: the first is consolidation therapy, which refers to the same or higher intensity of chemotherapy with chemotherapeutic drugs without cross-resistance as the chemotherapeutic drugs used for inducing remission therapy. Suitable for AML patients under 60 years of age, who are highly tolerated and can continue to receive 2–4 cycles of araC after remission. The other is to find a suitable bone marrow donor for bone marrow transplantation. Patients need a feasibility and risk assessment to decide on a bone marrow transplant. Bone marrow transplant patients need to remove AML cells before bone marrow transplantation, and not all patients can withstand this treatment, and there is a certain risk of surgical failure. Patients with AML may still relapse even with the most effective after remission therapy. Although most patients can achieve complete remission after chemotherapy, most of these patients will relapse with complications. In addition, approximately two-thirds of older patients (over 60 years old) with AML are refractory to standard chemotherapy and have a poor prognosis. Currently, the five-year overall survival in adult AML is only about 25% and about 65% in pediatric patients. Therefore, it is of great scientific and clinical importance to find new AML therapies, develop new therapeutic strategies to prolong survival in AML patients, and ultimately improve their cure rates.Acute myeloid leukemia (AML) is initiated by the abnormal myeloblasts accumulation, primarily in the bone marrow, which leads to bone marrow failure and finally causes death –39. InvoThe prognosis of AML patients ranges from the death of treatment to within days, i.e., treatment-related mortality to cure it, possibly. The main reason for patients is treatment resistance and often presents as relapse after remission rather than a TRM with decreasing incidence even in older patients. Knowledge of the various premutational states of gene therapy improves our ability to assign initial treatments. More importantly, whether ostensibly patients have measurable residual disease in time in remission should affect subsequent management. The US Food and Drug Administration has approved several new drugs, discussing their role in treatment.Based on data that indicate predictive covariates, the following factors are more important than the difference in acute myeloid leukemia—MDS disease, and some centers treated patients with blast number >10% as acute myeloid leukemia, even if they were treated by the WHO as a blast number of more than 2. From a biological perspective, the pattern of acute myeloid leukemia mutations occurring after previous hematological disorders  is generally more similar to that found in MDS than primary (new) acute myeloid leukemia.As research advances, many new therapies have been constantly proposed and applied to clinical experiments, such as small-molecule inhibitor-targeted therapies, immunotherapy. Among them, small-molecule inhibitor-targeted treatment is even more popular and highly concerned. Small molecule inhibitor-targeted treatment mainly suppresses cell growth, proliferation, and survival by targeting relevant cell signaling pathways  to achieve the effect of treating AML –47. At pTraditionally, new drugs have been tested in newly diagnosed cases of acute myeloid leukemia with relapse or at risk of disadvantage , 49–51. Finally, we may have overestimated our knowledge of targeted therapeutic targets. We note that the above beneficial effects of sorafenib, commonly considered as FLT 3 inhibitors, and, by adding to 7 + 3, appear to be the same in the population, regardless of whether they have FLT3 ITD or not. Therefore, there may have potential value once the outcome of targeted therapy. However, the limit is that there are not enough solid data that support the opinion, which suggest us to conduct further experiments to confirm this.


C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPRmt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPRmt to determine the relationship between hypoxic cell death, UPRmt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPRmt activation invariantly mitigated HIMPA. However, UPRmt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPRmt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in  Hypoxia, when extreme, will kill aerobic cells. The pathways whereby cells go from healthy to dead when they encounter hypoxia are many and complex. By the time cells are dying from hypoxia, virtually every energy dependent process in the cell is dysfunctional. Pathological processes that occur early in hypoxic exposure when the pathology may still be reversible are of particular interest. This pathologic time course certainly varies by cell type and organism. In mammals, central nervous system neurons and cardiac myocytes are exquisitely sensitive to hypoxia, and during complete ischemia begin dying in only a few minutes after dissolved oxygen is depleted , 2. On tMitochondria lie at the center of hypoxic biology and are logically the first organelle affected when oxygen levels fall below a crucial threshold. The vast majority of oxygen in metazoan cells is consumed by mitochondrial cytochrome C oxidase enabling the formation of ATP by ATP synthase. When oxygen levels fall, cytochrome C oxidase function fails, ATP levels decrease, and reactive oxygen species (ROS) are produced . BesidesC. elegans, we and others have identified mutants and RNAis that protect worms from hypoxic cellular and organismal injury  strain, confirmed that it was strongly hypoxia resistant  strongly reduced HIMPA as measured by UCR-11::GFP and TMRE ;daf-16(mu86) double mutant strain  on reducing aggregates is not explained by UPRmt activation  and rars-1(gc47) carry reduction of function mutations in threonyl-aminoacyl and arginyl-aminoacyl tRNA synthetases, respectively, and are strongly resistant to hypoxic death   Fig.  as previ52) Fig. . As was C. elegans is not commonly accompanied by UPRmt activation and that UPRmt activation is not always hypoxia protective. In other words, UPRmt activation is neither necessary nor sufficient for protection from hypoxic cell death in C. elegans. However, among RNAis selected solely for their ability to induce the UPRmt we did find a strong enrichment for hypoxia resistance, where five of the twelve UPRmt-activating RNAis conferred hypoxia resistance. By comparison, in a screen of over 16,000 RNAis, only 199 were found to reproducibly produce hypoxia resistance ) was obtained from the CGC. gcIs46 [ucr-11::GFP] and atfs-1(tm4919);gcIs46 were generated as previously described ) was used as a reporter for mitochondrial UPR activation [mt fluorescent reporter determinations were performed with worms exposed only to normoxic conditions. Briefly, a minimum of ten worms were mounted onto an agar pad on a standard microscope slide and imaged on a Zeiss axioskope 2 at ×10 magnification. Using ImageJ software (https://imagej.nih.gov/ij/), each worm was outlined and the fluorescence intensity within the area was measured and background intensity was then subtracted to obtain the total intensity/worm as previously described [SJ4100  and meclocycline (294 mM) in DMSO (Dimethyl sulfoxide) , were diluted to a final concentration of 100 μM with nuclease-free, deionized water and 300 µl was spread evenly onto a NGM plate seeded with OP50 onto which were placed synchronized eggs allowed to develop into 1-day-old adults. Plates containing TMRE  were made the same way as described above with 300 µl at a final concentration of 5 μM spread onto the plate. For TMRE staining, 1-day-old synchronized worms were transferred onto the TMRE-containing plates for 2 h before the plates were placed into the anaerobic chamber for 12 h for hypoxic exposure.Images of UCR-11::GFP and TMRE staining were acquired with confocal microscopy as previously described , 26. Brit-tests were used for statistical comparisons and all variances were of a similar magnitude except for death versus hypoxic exposure time curves  for simultaneous curve-fitting, t-tests, or Excel 2007 for t-tests . Values are expressed as mean ± SD of at least three independent experiments. A minimum p value of ≤0.05 was considered significant and was Bonferroni-corrected for multiple comparisons where appropriate. Sample sizes for technical replicates for number of animals was based on the variability from animal to animal for fluorescence-based assays and was always ≥10 or a minimum of 30 animals, where a percent of the population was scored. Based on our previous experience with hypoxic death population assays biological replicates were at least three per condition, and if significant variability was observed among replicates more were performed until clear reproducibility was achieved. In terms of randomization, strains were not randomized as they are genetically identical and raised under environmentally identical conditions.Two-sided unpaired or paired, as appropriate, ves Fig. , where sTable S1Table S2Figure S1Figure S2Supplementary Figure Legends