BRCA1/2 or PALB2, both first-line and maintenance therapy differs significantly. These mutations confer a loss of double-strand break DNA homologous recombination (HR), substantially altering drug sensitivities. In this review, we discuss the current treatment guidelines for PDAC tumors deficient in HR, as well as newly emerging strategies to improve drug responses in this population. We also highlight additional patient populations in which these strategies may also be effective, and novel strategies aiming to confer similar drug sensitivity to tumors proficient in HR repair.For the small subset of pancreatic ductal adenocarcinoma (PDAC) patients with loss-of-function mutations to BRCA1/2, PALB2, and other functionally related genes. Recent evidence suggests that the presence of such mutations in pancreatic ductal adenocarcinoma (PDAC), the most common and lethal pancreatic cancer histotype, significantly alters drug responses both with respect to first-line chemotherapy and maintenance therapy. In this review, we discuss the current treatment paradigm for PDAC tumors with confirmed deficits in double-strand break HR, as well as emerging strategies to both improve responses to PARP inhibition in HR-deficient PDAC and confer sensitivity to tumors proficient in HR repair.Pancreatic cancer is projected to become the second leading cause of cancer-related death by 2030. As patients typically present with advanced disease and show poor responses to broad-spectrum chemotherapy, overall survival remains a dismal 10%. This underscores an urgent clinical need to identify new therapeutic approaches for PDAC patients. Precision medicine is now the standard of care for several difficult-to-treat cancer histologies. Such approaches involve the identification of a clinically actionable molecular feature, which is matched to an appropriate targeted therapy. Selective poly (ADP-ribose) polymerase (PARP) inhibitors such as Niraparib, Olaparib, Talazoparib, Rucaparib, and Veliparib are now approved for several cancers with loss of high-fidelity double-strand break homologous recombination (HR), namely those with deleterious mutations to  BRCA1, BRCA2, or PALB2 [Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death in the United States, with an overall survival rate of 10% . Though or PALB2 ,6. Patieor PALB2 .BRCA-mutated tumors, including breast, ovarian, prostate, and now pancreatic cancers [BRCA1, BRCA2, and PALB2, both first-line and maintenance therapy differ significantly [This approach is based on the ability of PARP inhibitors to limit the capacity for single-strand break repair, leading to the accumulation of DNA damage and eventually cell death in the setting of genetic HR deficiency (HRD) ,9. Accor cancers ,10,11,12ATM, leading to increased genetic instability due to a loss of high-fidelity double-strand break HR and dysregulation of cell cycle checkpoints [Atm in mice accelerates tumor development, increasing genomic instability and enhancing metastasis [As with many cancers, there is a strong genetic component to PDAC. Recent estimates suggest that as many as 10% of PDAC cases can be attributed to a familial predisposition . Accordickpoints . Consisttastasis ,21.BRCA1 and/or BRCA2 also confers an increased risk of PDAC, again due to the presumptive loss of double-strand break HR [Brca2 heterozygosity cooperated with oncogenic KRAS to promote PDAC development [Trp53 signaling must be modified prior to Brca2 inactivation for Brca2-deficient cells to form tumors [PALB2 increase PDAC risk [PALB2 in PDAC biology. However, clinical data strongly suggest that patients with a known or family history of these genetic events may benefit from early screening [Hereditary breast and ovarian cancer (HBOC) syndrome caused by deleterious mutations in break HR . This iselopment , with sum tumors . SimilarDAC risk , though creening ,27.ATM, BRCA1/2, and PALB2 mutations are relatively uncommon in PDAC. In the case of ATM, the rates are highly varied with somatic mutations identified in 2–18% of PDAC tumors and germline mutations identified in 1–34% [BRCA and PALB2, germline mutation frequencies are estimated to be present in 5–9% of patients [N = 741) as described previously [ATM, 1.08% for BRCA1, 1.48% for BRCA2, and 0.54% for PALB2  pathogenic or potentially pathogenic mutations. Four of these mutations were found in non-BRCA genes, with two affecting ATM, one affecting PALB2, and one affecting RAD50 [BRCA1, BRCA2, PALB2, ATM, ATR, CHEK2, RAD51, and FANC. Using this expanded panel, HRD prevalence ranged from 14.5–16.5% using targeted next-generation sequencing, and 24–44% via whole-genome or whole-exome sequencing [Additional genomic studies have offered varying mutation frequencies for genes involved in DNA HR, particularly when including non-classic genes that may be associated with HRD. For example, a study of 60 PDAC patients already tested for ng RAD50 . Consistng RAD50 , with sing RAD50 . Accordiquencing . Hence, Chemotherapy is the backbone of treatment for both localized and disseminated PDAC . For theBRCA1/2 or PALB2 mutations. This is based on the longstanding hypothesis that tumors with these and related mutations have a loss of high-fidelity double-strand HR and have improved therapeutic responses to platinum-based chemotherapy [BRCA1-, BRCA2-, or PALB2-mutated PDAC displayed a 58% response rate to platinum-based chemotherapy compared to 21% of controls [BRCA1/2- or PALB2-mutated PDAC, front-line therapy varies significantly.However, current guidelines support the use of alternate treatment regimens for the 5–9% of PDAC patients with deleterious otherapy ,44, a photherapy ,46. Speccontrols . Hence, BRCA-mutated PDAC are more likely to achieve pathologic complete responses to FOLFIRINOX, which the authors attributed to improved sensitivity to the platinum-agent, Oxaliplatin [BRCA- or PALB2-mutated PDAC, which led to encouraging 2- and 3-year survival rates of 31% and 18%, respectively [BRCA1/2 or PALB2, the current treatment guidelines support either FOLFIRINOX or the combination of Gemcitabine and Cisplatin .A recent study has demonstrated that patients with borderline resectable, liplatin . Additioectively . Hence, BRCA1/2 or related mutations. Though results are not yet available, FOLOX-A will be compared to Gemcitabine and nab-Paclitaxel, with a primary endpoint of disease progression during neoadjuvant therapy [Importantly, the ongoing phase 2 PRIMUS-001 (ISRCTN75002153) and PRIMUS-002 (ISRCTN34129115) trials are also exploring the FOLOX-A regimen  in PDAC patients harboring  therapy ,49BRCA mutation [BRCA-mutated PDAC. This study enrolled 16 patients with stage 3/4 disease, 14 of which had received prior platinum-based chemotherapy. Five had a known BRCA1 mutation, and the remaining 11 had a known BRCA2 mutation. In this cohort, no confirmed partial responses were observed, four patients showed stable disease, and 11 had progressive disease. Six patients had a grade III toxicity, most commonly fatigue [As mentioned, PARP inhibitors are an important part of the treatment for cancers deficient in DNA HR. This is based on longstanding observations that PARP inhibitors impair single-stranded break repair, promoting synthetic lethality in the setting of HRD . Accordimutation . For exa fatigue .BRCA-mutated patients who had not progressed during first-line platinum-based chemotherapy. The 154 patients enrolled were randomized into two groups, with 92 receiving Olaparib and 62 a placebo. The Olaparib-treated group demonstrated a significant improvement in progression-free survival (7.8 months compared to 3.8 months on placebo), though an interim analysis did not show a statistically significant increase in overall survival. Olaparib was well tolerated in this study, with no significant difference in health-related quality of life between groups despite the higher rate of grade 3 or worse adverse events in the Olaparib group, most commonly in the form of fatigue, nausea, or other gastrointestinal symptoms [BRCA-mutated PDAC in 2019.A subsequent phase 3 trial (POLO) has evaluated the PARP inhibitor Olaparib as maintenance therapy, specifically for BRCA/PALB2-mutated PDAC. An ongoing phase 2 study is exploring single-agent Rucaparib in patients with germline or somatic BRCA1-, BRCA2-, or PALB2-mutated PDAC that received at least 16 weeks of platinum-based chemotherapy without evidence of chemo-resistance. Of the 42 evaluable patients thus far, the overall response rate was 41.7%, which translated to a median progression-free survival of 13.1 months, and a median overall survival of 23.5 months. Importantly, no new safety concerns were noted [The PARP inhibitor Rucaparib has also shown promise for re noted .BRCA-mutated and BRCA-non-mutated PDAC. This approach was particularly effective in the BRCA-mutated group , leading to a phase 2 study in exclusively BRCA-mutated patients [BRCA1/2 or PALB2. In patients with HRD, the overall response rate was 57% [PARP inhibition has also been evaluated in combination with chemotherapy in PDAC patients. For example, a phase 1 study evaluated the combination of Cisplatin, Gemcitabine, and Veliparib in both patients . Howeverpatients . Additio was 57% .Several ongoing trials are also exploring PARP inhibitors in PDAC patients, both as monotherapy and in combination with other treatments  is a serine/threonine-protein kinase that acts as a central regulator of cell cycle checkpoints and HR . Mechaniignaling . Accordiignaling , and ATRpathways .ATM-mutated tumors showing early promise. In brief, 25 patients with confirmed HRD or other DNA-repair deficiency received both Ceralasertib and Olaparib. Though only 8.3% of patients demonstrated responses by RECISTv1.1 criteria, 62.5% derived a clinically meaningful benefit from treatment. This approach was well tolerated, with 32% of patients experiencing a grade 3 or worse toxicity in the form of anemia, neutropenia, or thrombocytopenia [Consistent with these observations, the ATR inhibitor BAY 1895344 cooperated with Olaparib in in vivo models of breast and prostate cancer , as did BRCA-wild type triple negative breast cancer (TNBC) cells to PARP inhibitors, overcoming both acquired and de novo drug resistance [BRCA1-mutated breast cancer [In HR-proficient PDAC, preclinical data support the combination of Olaparib, the ATR inhibitor VE-822, and the dual mTOR kinase/DNA-PK inhibitor CC-115 as maintenance therapy after platinum-based chemotherapy . This apsistance . Similart cancer . Recentlt cancer .BRCA-mutated tumor cells, and was able to overcome acquired resistance to PARP inhibition when combined with the cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine [Other targets have also been suggested, notably DNA polymerase Polθ (Polθ). Pharmacologic inhibition of Polθ via ART558 induced DNA damage and synthetic lethality in BRCA-mutated tumor cells, and synergized with PARP inhibition . Though yuridine . Similaryuridine . In highyuridine . Hence, BRCA1 and RAD51 [In addition to strategies targeting DNA repair processes directly, a number of epigenetic inhibitors are also being explored with PARP inhibitors in cancer, including Bromodomain and Extra-Terminal motif (BET) inhibitors. BET proteins include BRD2, BRD3, BRD4, and BRDT ,72,73. Tnd RAD51 . Similarnd RAD51 , furthernd RAD51 ,85, the BRCA1 mutation [BRCA-mutated phenotype and enhancing the tumoricidal effects of PARP inhibitors [In addition to BET inhibitors, other epigenetic inhibitors are also showing early promise, many of which directly target posttranslational histone modification. For instance, a therapeutic inhibition of Histone Deacetylases (HDACs) is emerging as an additional means of disrupting homologous recombination, largely by reducing the expression of key HR genes such as RAD51 ,87,88,89mutation . Similarmutation . As studhibitors ,93,94, thibitors .BRCA-wild type breast cancer cells, showing synergistic tumor cytotoxicity [BRCA-status [BRCA mutation, and increased sensitivity to both PARP inhibition and ionizing radiation [In addition to HDAC inhibitors, select inhibitors of DNA methyltransferases (DNMTs) are also showing potential as a means of improving responses to PARP inhibition. A seminal study explored combined DNMT and PARP inhibition in acute myeloid leukemia (AML) and toxicity . In ovarA-status . Similaradiation . Hence, adiation , this isBRCA mutation. However, 27.3% of patients in the combination arm experienced a grade 3 or worse adverse event, most commonly diarrhea or hypertension. Hence, the authors recommended that investigations should include assessments of quality of life due to the side-effect profile. [KRAS-mutated colorectal cancer cells [Several additional combination strategies are also showing promise. As mentioned, recent studies have explored the combination of VEGF and PARP inhibitors. Following a promising phase 1 trial , a landmprofile. ,102. Simer cells , though BRCA2 mutation. Accordingly, the ALDH1A1 inhibitor NCT-501 synergized with Olaparib in cell culture and xenograft models of BRCA2-mutated ovarian cancer [Other proposed targets include Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), which has very recently been linked to microhomology-mediated end joining (MMEJ) and resistance to PARP inhibition. In brief, in ovarian cancer cells, Olaparib increased ALDH1A1 expression through the BET protein BRD4, thereby activating alternate DNA repair pathways in cells harboring a n cancer . Similarn cancer .PIK3CA-mutated ovarian cancer cells, BKM120 cooperated with Olaparib, in part through downregulation of BRCA1 [PIK3CA-wild type cells [Several studies are also exploring the effects of the PI3K pathway on responses to PARP inhibitors. The intersection between PI3K signaling and HR has long been of interest, as PTEN-deficient tumors have been suggested to have reduced expression of RAD51, thereby conferring improved sensitivity to PARP inhibition. Though encouraging, this is rather controversial and PTEN status is not generally considered a useful predictor for PARP inhibitor sensitivity at this time ,108,109.of BRCA1 , with sipe cells . In HR-ppe cells . In earlpe cells . Hence, pe cells .BRCA status [Other potential candidates for therapy have also been proposed including MYC. Selective MYC inhibition has been demonstrated to sensitize TNBC cells and other aggressive MYC-overexpressing tumors to PARP inhibition independent of A status . In precA status . As MYC A status ,119, MYCSeveral other targets for therapy have been suggested. Notable examples include Glycogen synthase kinase 3β (GSK3β), as a high throughput screen of 99 anti-cancer compounds determined that GSK3β inhibition cooperated with PARP inhibitors in colon cancer, suppressing HR and increasing both replication stress and DNA double-strand breaks . SeveralBRCA1/2 or PALB2 mutations, the current treatment paradigm differs significantly from that for other patients, with the combination of Gemcitabine and Cisplatin as an appropriate first-line treatment option [BRCA- and PALB2-mutated PDAC tumors have distinct molecular characteristics that may pave the way for novel combination strategies that have yet to be evaluated. For example, a recent study evaluated 2818 PDAC specimens using a combination of next-generation sequencing and immunohistochemistry. Though BRCA and PALB2 mutations were relatively uncommon, tumors from BRCA- and PALB2-mutated patients had distinct mutational profiles compared to non-mutated patients, were more likely to be PD-L1 positive, and had a comparatively high tumor mutational burden (TMB). The authors, therefore, concluded that these data provided rationale to evaluate combined PARP and immune checkpoint inhibition in BRCA/PALB2-mutated PDAC patients [There is currently no effective treatment for PDAC. For patients with confirmed HRD in the form of deleterious t option  and the t option . Though patients . This appatients , prostatpatients  and lungpatients . As mentpatients , though BRCA and PALB2 mutations are not the sole predictors of either HRD or PARP inhibitor sensitivity [BRCA-mutated TNBC patients as more likely to respond to platinum chemotherapy [BRCA1, BRCA2, ATM, FANCA, CHEK2, PALB2, NBN, or HDAC2. This identified an HRD frequency of 33%, with 88% of biomarker-positive patients demonstrating therapeutic responses to Olaparib [BRCA-mutated tumors, and were observed in several patients with mutations to other HR genes including ATM, NBN, RAD51C, and RAD51D [BRCA and PALB2 mutations as the defining criteria [Additionally, there is now consensus that sitivity . For exaotherapy . In metaOlaparib . Similard RAD51D . In PDACcriteria ,38,39. Hcriteria ,141,142.It is also important to note that there is mounting evidence that HR-proficient PDAC patients may also benefit from PARP inhibitors when combined with additional strategies targeting either HR repair processes, both directly and indirectly. This may prove to be an effective strategy to enhance therapeutic responses in PDAC patients, though such approaches have yet to enter clinical evaluation. It is also important to note that clinical responses to PARP inhibitors are not solely driven by HRD. In other cancers, the efficacy of these medications involves several other factors including replication, oxidative, and ER stress ,146,147.BRCA/PALB2 mutations are rare in PDAC, this raises the important issue of whether it is justifiable to sequence all tumors given the expense [BRCA and similar mutations have been limited to PDAC patients with the relevant family history.Finally, while precision medicine can offer significant benefit to select PDAC patients and can even facilitate the testing of at-risk relatives, there are several additional factors that must also be considered. As  expense . Though BRCA1/2 testing remains underutilized in cancer patients, even for those with insurance coverage and access to specialty genetic services [However, cost is seemingly not the only barrier to testing, as services . The reaservices . Hence,


Genetic manipulation of GABA metabolism and receptors, respectively, reveal positive relationships between GABA levels and abiotic/biotic stress tolerance, and between malate efflux from the root and heavy metal tolerance. The application of exogenous GABA is associated with lower ROS levels, enhanced membrane stability, changes in the levels of non-enzymatic and enzymatic antioxidants, and crosstalk among phytohormones. Exogenous GABA may be an effective and sustainable tolerance strategy against multiple stresses under field conditions.Global climate change and associated adverse abiotic and biotic stress conditions affect plant growth and development, and agricultural sustainability in general. Abiotic and biotic stresses reduce respiration and associated energy generation in mitochondria, resulting in the elevated production of reactive oxygen species (ROS), which are employed to transmit cellular signaling information in response to the changing conditions. Excessive ROS accumulation can contribute to cell damage and death. Production of the non-protein amino acid γ-aminobutyrate (GABA) is also stimulated, resulting in partial restoration of respiratory processes and energy production. Accumulated GABA can bind directly to the aluminum-activated malate transporter and the guard cell outward rectifying K T. TArabidroxisome .Kms in the low micromolar range) as substrates to produce GABA, β-alanine and γ-butyrobetaine, respectively [ataldh10A8,9 seedlings are phenotypically normal, and the GABA and Glu levels are similar across the mutants and WT [Early support for the existence of ABAL/pyrroline dehydrogenase in plants is available from the production of radiolabeled GABA from exogenously supplied radiolabeled Put, and the suppression of GABA production by the addition of aminoguanidine, a CuAO inhibitor ,48. Moreectively ,51. Theys and WT .1-pyrroline-5-carboxylate synthetase and NADP-dependent ∆1-pyrroline-5-carboxylate reductase [1–pyrroline/ABAL and oxidized to GABA via ABAL/pyrroline dehydrogenase activity. To date, there is no direct evidence for the contribution of proline to GABA production in planta.Proline is another potential indirect source of cytosolic GABA . It is deductase . Prolineeductase . Pyrroliatgabp mutant is not eliminated, suggesting the possible existence of other mitochondrial GABA carriers with overlapping or redundant functions [Km Spd = 55 μM, Km Put = 85 μM, Km arginine = 1.4 mM [The mitochondrial-localized bidirectional amino acid transporter/GABA permease (AtBAT1/GABP) links the anabolic and catabolic portions of the GABA shunt  Figure . Howeverunctions . The subunctions ; GABA, bunctions ; Km Spd = 1.4 mM ).Kms for GABA, pyruvate and glyoxylate of 0.18−0.34 mM, 0.14 mM and 0.11 mM, respectively [AtGABA-TOG gene has not yet been identified. In our opinion, a recent paper describing sugarcane GABA-TOG activity lacks rigor [atgaba-tp mutant is phenotypically normal, except for lower seed production, and the leaf GABA level can increase up to 16-fold without an effect on the Glu level [The pyruvate- and glyoxylate-dependent GABA-T (GABA-TP) catalyzes conversion of GABA to SSA in the mitochondrion ,58,59 F. It is rectively ,58. Thisectively ,61,62; hks rigor , and theks rigor . Furtherks rigor ,65,66. Tlu level ,68,69,70Km SSA = 15 μM, Km NAD = 130 μM, Ki NADH = 122 μM, Ki ATP = 8 mM). The atssadh mutant overaccumulates GABA (28 nmol g−1 FM) and H2O2 by 2- and 4-fold, respectively [The conversion of SSA into succinate in the mitochondrion is catalyzed by SSADH activity ,72 Figu. The AtSectively ,74. Succectively ,28,74. Nectively ,76. Thes+ (Ki = 1–3 μM) [atglyr1,2 mutants and NAD kinase 1 overexpression line accumulate less and more GHB, respectively, with submergence than the WT [Arabidopsis lines with altered GLYR activity responds differentially to SSA or glyoxylate under chilling conditions [2, and suggest that AtGLYR1,2 are part of an adaptive response to stress-induced changes in redox balance [osglyr1/2 double mutant displays stunted growth under photorespiratory conditions, compared to the WT [SSA can be reduced to γ-hydroxybutyrate (GHB) via NADPH-dependent glyoxylate/SSA reductases in the mitochondrion/plastid (AtGLYR2/SSR2) and cytosol (AtGLYR1/SSR1) ,81,82,83 1–3 μM) ,85. The n the WT . The groo the WT , validato the WT . The twoo the WT ,16.Succinate is converted to citrate and 2-OG in the TCAC ,28 Figu. Two potGlu availability is an essential regulator of GAD activity, and the generation of cytosolic Glu from succinate bypasses two reactions of the TCAC . However, it does not exclude participation in the cytosolic GAD reaction of Glu originating in the plastid via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle  Figure . The movArabidopsis grows efficiently on GABA as the sole N source, providing the first strong evidence for GABA uptake by plant cells [Km = 12.0 and 1.7–5 mM, respectively [Km = 10 μM) [atgat1 mutant is unaffected by the addition of exogenous GABA, but it increases in the WT, confirming that AtGAT1 plays a role in GABA influx into the cell [nt cells  . AtPectively ,97. AtGA= 10 μM) ,99. EndoGABA is released from asparagus mesophyll cells via an unknown mechanism , but eviTo date, two organellar transporters for GABA have been described. The mitochondrial AtGABP was the first see . AnotherArabidopsis plants. Subsequently, GABA declines, but succinate remains steady for 72 h. GHB accumulates sharply from 24 to 48 h and then declines to 96 h. Glu accumulates in a linear fashion from 12 to 96 h, whereas Put does so from 24 h. Furthermore, Espinoza et al. [Kaplan et al.  showed ta et al.  demonstrSome generalizations are possible from these two studies: (i) the cold-induced accumulation of GABA, alanine, succinate and GHB are not correlated with the availability of Put and proline; (ii) succinate and GHB may be concomitantly generated from SSA; (iii) SSA and succinate turnover may be restricted or an alternate source of succinate exists; and, (iv) Glu/Gln metabolism is altered under cold conditions. A definitive explanation for the increasing accumulation of Glu is not possible, despite the accumulation of three well-known products of Glu metabolism , though protein hydrolysis is known to be stimulated by low temperature .Glycine max (L.) Merr.) roots by 11- to 17-fold, compared to the control, as well as the diamine oxidase activity by 52–86%, but decreases the levels of Put, Spd and Spm [−1 fresh mass (FM), but decreases the GABA level from 10.8 to 6.6 μmol g−1 FM. While these findings could be interpreted as support for the derivation of GABA from Put [Treatment with 50–150 mM NaCl for 6 d increases the GABA level in soybean  cells are 1.2, 2.0, 0.12, 0.84 and 6.77 μmol g−1 FM, respectively, over a 2-d period, whereas they are 13.0, 6.26, 31.2, 1.22 and 2.71 μmol g−1 FM, respectively, in cells adapted to water stress (25% polyethylene glycol 6000) [15N-labeling kinetics reveals that adaptation to water stress increases the N flux into GABA and alanine, suggesting high pyruvate availability and rapid turnover of both amino acids. The rate of GABA synthesis and catabolism, respectively, are 0.80 and 0.785 μmol h−1 g−1 FM in control cells, and 2.4 and 1.26 μmol h−1 g−1 FM in adapted cells. About 76% of the GABA is located in a metabolically inactive pool in unadapted cells, but only 38% in adapted cells. The proline pool increases by 300-fold due to greater synthetic rates from Glu and restricted oxidation, and the metabolically inactive Gln storage pool becomes depleted. Notably, the rate of nitrogen assimilation doubles, even though the total soluble protein (on a dry mass basis) decreases by 30%.Other studies have modeled the stress-induced changes in flux through the GABA shunt using suspension cultures. For example, the mean GABA, alanine, proline, Glu and Gln pools in control tomato (ol 6000) . ComputeVigna unguiculata (L.) Walp) cells at 26 °C are 0.27, 5.37, 0.42, 1.79 and 4.11 μmol g−1 FM, respectively [−1 FM after 2 h and 1 d, respectively, and the other amino acids also accumulate, but less extensively than GABA. Total free amino acid levels increase approximately 1.5-fold after 1 d at 42 °C. The computer simulation suggests that heat shock induces a 63-fold increase in the rate of GABA synthesis over the first 2 h, without any change in the rate of GABA catabolism. The rate of GABA synthesis over the next 22 h increases 7-fold, and this is accompanied by a 3-fold increase in GABA catabolism. The rates of alanine and proline synthesis increase by 0.6 and 1.7-fold, respectively, over the 1-d period. The size of the free amino acid pool increases within 1 d, and the rate of protein synthesis decreases by 20–30%. An accelerated rate of protein degradation may also contribute to the effects of heat shock on the amino acid perturbations [In related research, the mean GABA, alanine, proline, Glu and Gln pools in unadapted cowpea  [2+/CaM complex or H+ in the cytosol. The GOGAT- or GDH-mediated regeneration of Glu from 2-OG in the plastid and mitochondrion, respectively, is sufficient to sustain the increased formation of cytosolic GABA for a limited duration. However, the relative importance of PAs and proline to GABA accumulation remains controversial [The stress-activated acceleration of flux through the GABA shunt can be explained by increases in GAD activity and GABA synthesis due to elevated levels of Glu  ,110,111,oversial .2O2 level supports this interpretation. Che-Othman et al. [Recent studies have assessed the interaction between GABA catabolism and respiration. For example, increases in GAD activity and GABA level (1-fold) in leaves of tomato plants after 5 d of exposure to 200 mM NaCl, together with the 25% decrease in succinate , suggestn et al.  also foun et al. .+ is regenerated via fermentation reactions [+ required for oxidation of 2-OG is apparently produced by the anti-clockwise operation of the TCAC malate dehydrogenase, which increases malate accumulation. The carbon flux from SSA to succinate is not eliminated, even though the NADH/NAD+ ratio is presumably altered to some degree. GABA accumulates, at least in part, due to the stimulation of GAD activity by bound Ca2+/CaM or lower cytosolic pH. Overall, both GABA and succinate appear to be temporary storage metabolites that readily supply the TCAC when hypoxia is mitigated [Respiration in soybean roots is reduced by 40%, after 6 h of hypoxia, but the ATP and pyruvate supply is enhanced via an activated glycolytic pathway, and the cytosolic NADeactions . The diritigated .+ ratio might be attributed to increases in the activities of NAD kinases [2O2 [2 fixation, leading to the underutilization of NADPH, over-reduction of the photosynthetic electron transport chain, and the generation of ROS. Stomatal closure also increases Rubisco activity, leading to the glycolate oxidase-mediated generation of H2O2 [The hypoxia-induced increase in NADPH/NADP kinases  and the  kinases . In addi kinases , NADPH-m kinases ,83, and ses [2O2 ,117. Wit of H2O2 .atgad2 single and atgad1/2 double mutants of Arabidopsis do not accumulate GABA in response to salinity, drought or hypoxia, but they become hypersensitive to the stress [ataldh10A8,9 single mutants have been shown to decrease the GABA level and confer a hypersensitive salinity phenotype [atgaba-t mutant increases with salinity or hypoxia and the phenotype is more tolerant to the stress [atgad1/2 mutant results in more RBOHF/NADPH oxidase-mediated H2O2 production and K+ efflux via the GORK and Shaker-type outward rectifying K+ channels than in the atgaba-t mutant [atgaba-t mutant doubles with salinity (28 μmol g−1 DM), while the succinate and 2-OG levels decrease or are unchanged, but the mutant becomes hypersensitive to salinity [atssadh mutant overaccumulates GABA, H2O2 and GHB in the absence of stress, and displays hypersensitivity to heat stress [e stress ,70,118. henotype . In contt mutant ,118. Sursalinity . The atst stress ,74.SlGAD1-4 prevents the salinity-induced GABA accumulation and confers a hypersensitive salinity phenotype, whereas knockdown of SlGABA-T1-3 slightly increases the GABA level, while conferring a hypersensitive salinity phenotype [SlSSADH increases the GABA level and confers a salinity tolerant phenotype. Furthermore, the GABA and H2O2 levels are higher and lower, respectively, than the corresponding levels in the SlGAD1-4 and SlGABA-T1-3 knockdown lines . Thus, the tolerance is inversely related with the H2O2 level.Knockdown of tomato henotype  -tolerant phenotype, greater K+ retention and hypoxia-induced Ca2+ signaling, and does not show any change in K+ efflux in response to GABA [+ from root cells [Wheat root TaALMT1 is responsible for malate efflux, but this is inhibited by GABA binding to the cytosolic surface of the protein ,101,102  to GABA ,117  level upregulates GORK activity, resulting in stomatal closure and drought tolerance. The opening of AtALMT6 and AtALMT4 mediates malate efflux from the vacuole. Various Ca2+-permeable channels contribute to the elevation of cytosolic Ca2+ during stress [ALMTs and GORK facilitate the functioning of stomata  Figure . During g stress ,122.Arabidopsis mutants with altered GABA levels. The atgad1/2 double mutant has impaired stomatal closure and a drought-susceptible phenotype [atgad2 and atalmt9 mutants demonstrated that the drought-induced accumulation of GABA suppresses light-induced stomatal opening, whereas it has no effect under constant light [almt12 mutant showed WT sensitivity to GABA, whereas dark-induced stomatal closing is insensitive to GABA [A role for GABA accumulation in drought resistance has been shown using henotype  (Table 1nt light  (Table 1 to GABA  (Table 2 to GABA  (Table 23+) ions in the soil solution under low pH limits plant growth and productivity [3+ tolerant (ET8) and sensitive (ES8) lines of wheat. In the absence of Al3+, GABA has no effect on malate efflux under acidic conditions. However, malate efflux increases in response to Al3+ treatment, and decreases in response to Al3+ and exogenous GABA in ET8, but not in ES8. While the GABA level is higher in ET8 than in ES8 under acidic conditions, Al3+ reduces the GABA level in both lines to similar levels. This suggests that high endogenous GABA inhibits the activity of TaALMT1 in the absence of Al3+, and that both malate and GABA are exported from the cytosol when TaALMT1 is Al3+ activated, resulting in the sequestration of Al3+ and modulation of the plant sensitivity to Al3+ [3+ treatment under acidic conditions [3+ [The accumulation of free aluminum  , which cPlants must endure a wide variety of abiotic and biotic stresses under field conditions. To prevent significant yield losses, many crop improvement programs strive to develop stress-tolerant cultivars. Plants may respond uniquely to different or simultaneous stresses, so breeding tolerance against one stress may be at the expense of tolerance to another. With climate change, plants will likely experience more extreme weather events or multiple stresses, including plant diseases. It would therefore be beneficial to develop a tolerance strategy against multiple stresses.2, heavy metal toxicity) and biotic  stresses  are extensively employed to extend the postharvest life of horticultural commodities, especially botanical fruits. However, these crops may suffer from chilling injury and other physiological disorders, as well as fungal decay. The use of exogenous GABA to improve the marketability of stored horticultural commodities will be described in a companion paper.Low temperature and controlled atmosphere conditions (low O


TYK2) as an oncogene that is frequently mutated or overexpressed in many types of cancer and metastases. A member of the Janus kinase (JAK) family, TYK2 mediates the signals of numerous cytokines involved in immune and inflammatory signaling. In cancer cells, activation of TYK2 can lead to decreased cell death as well as increased cell growth and invasion. Multiple drugs that specifically block TYK2 or JAKs are currently FDA-approved or in clinical trials. In this review, we provide an overview of the screening, molecular, and animal studies that have characterized the role of TYK2 in cancer and metastases, and the potential of TYK2 inhibitors as effective cancer therapies. Cancer deaths are predominantly due to metastases rather than the primary tumors, and thus there is an urgent need for the discovery of more effective drug therapies for metastatic cancer. Recent genomics, transcriptomics, and proteomics studies have identified tyrosine kinase 2 (TYK2) as an oncogene promoting progression and metastases of many types of carcinomas, sarcomas, and hematologic cancers. TYK2 is a Janus kinase (JAK) that acts as an intermediary between cytokine receptors and STAT transcription factors. TYK2 signals to stimulate proliferation and metastasis while inhibiting apoptosis of cancer cells. This review focuses on the growing evidence from genomic and proteomic screens, as well as molecular studies that link TYK2 to cancer prevalence, prognosis, and metastasis. In addition, pharmacological inhibition of TYK2 is currently used clinically for autoimmune diseases, and now provides promising treatment modalities as effective therapeutic agents against multiple types of cancer.Advances in genomic analysis and proteomic tools have rapidly expanded identification of biomarkers and molecular targets important to cancer development and metastasis. On an individual basis, personalized medicine approaches allow better characterization of tumors and patient prognosis, leading to more targeted treatments by detection of specific gene mutations, overexpression, or activity. Genomic and proteomic screens by our lab and others have revealed tyrosine kinase 2 ( Revolutionary advancements in bioinformatics techniques and computational data analysis within the last decade have transformed modern cancer diagnostics and therapeutics . InnovatTYK2) mutations, fusion proteins and expression changes in a variety of hematological cancers, carcinomas and soft-tissue sarcomas . Ac. AcJAK2, domains . In pedileukemia ,24. Anotleukemia ,26. Furtleukemia .Additionally, four TYK2 fusion proteins have been identified in several hematologic cancers using genomic and transcriptomic sequencing ,19,25. ITYK2 in malignancy is complex and likely is cell type and context dependent. In some settings, TYK2 appears to play a role in suppressing tumor growth, and a few studies have reported lower TYK2 expression or loss-of-function (LOF) mutations associated with cancer development or progression .,52.47,52In a negative feedback loop, STATs induce transcription of suppressor of cytokine signaling (SOCS) genes . SOCS1-7The earliest studies on TYK2 signaling found it to be an intermediate in the IFNα and IFNβ pathways  66]. Su. Su66]. TYK2 also associates with the IL-10R2 chain which, in combination with the JAK1-associated IL-28R chain, mediates signal transduction of type III IFNs through STAT1/STAT2 heterodimers and IRF9 ,76. The The IL-12 family and the IL-10 family of cytokines also utilize TYK2 signal transduction in a balance of immunomodulatory and inflammatory responses. Members of the IL-12 family (IL-12 and IL-23) bind to JAK-associated IL-12Rβ2 or IL-23R chains, respectively, which heterodimerizes with the TYK2-associated IL-12Rβ1 chain to activate primarily STAT4 and STAT3, respectively  50]. ST. ST50]. IL-10 family members are essential anti-inflammatory cytokines with immunosuppressive actions that promote epithelial homeostasis and barrier function in response to infection or inflammatory conditions, while IL-10 deficiency is involved in autoimmune disorders . HoweverOther cytokines that signal through TYK2 include those that bind to the gp130 receptor subunit, e.g., IL-6, IL-11, IL-27, oncostatin M (OSM), cardiotrophin-1 (CT-1), and leukemia inhibitory factor (LIF). However, TYK2 is redundant with other JAKs in these pathways. A full list of cytokines and receptors reported to signal through TYK2 have been reviewed previously ,47.TYK2 as a susceptibility gene in auto-inflammation, immunodeficiency and other immune responses as well as in cancer . I. I105]. survival . Likewissurvival . TYK2 desurvival . In esopsurvival . The flasurvival . Fibroblsurvival . In B-cesurvival . Similarof BCL-2 . In headof BCL-2 .STAT transcription factors have also been widely studied for their role in carcinogenesis, as they are frequently mutated or highly activated in cancer . The majIn some normal and cancer cell types, TYK2 is involved in IFN signaling to induce apoptosis through pro-apoptotic proteins ,121,122.TYK2 has emerged as an oncogene that promotes migration, invasion and metastasis in multiple types of cancer, including stomach, MPNST, breast cancer, liver cancer, colon cancer, prostate cancer and lung cancer [Metastasis is the primary driver of cancer mortality. Thus, it is imperative to understand the molecular mechanisms underpinning metastasis to reduce cancer deaths . Epithelg cancer ,115,116.TYK2 and JAK3 mRNA expression were positively associated with tumor grade, stage, and lymph node metastasis status, and patients with high TYK2 expression in their tumors had shorter overall survival [TYK2 SNP , although the mechanism of this mutation was not investigated [TYK2 mutations in MPNST [Tyk2 decreased MPNST tumor burden and increased overall survival [Using an RNA-seq bioinformatics approach to compare stomach adenocarcinoma and normal tissues, survival . Therefosurvival . A genomstigated . Likewissurvival .TYK2 gene expression by shRNA decreases invasion through decreased urokinase-type plasminogen activator (uPA), which is involved in the activation of MMPs that degrade extracellular matrix proteins [The process of invasion is critical in EMT and metastases, and is regulated in part by tight junction proteins and matrix metalloproteinases (MMP) . In lungproteins ,127. Annproteins . Knockdoproteins .As discussed earlier, the role of TYK2 in cancer is complex, and some groups have reported an inhibitory role of TYK2 in metastases ,42,128. Selective inhibitors of JAKs (JAK1-3/TYK2) were originally developed as therapeutics for autoimmune diseases , as well as organ transplantations, due to their ability to suppress the immune response  129,130130. In 2In an effort to lessen side effects while improving efficacy, TYK2 inhibitors (TYKinibs) have become a recent focus in drug discovery ,148,149.JAK2 kinase domain that reduce drug binding, or to activation of the RAS, ERK and Akt pathways [JAK2 V617F [MYB-TYK2 fusion protein eventually become insensitive to the JAK/TYK inhibitor cerdulatinib after prolonged incubation due to a mutation in the TYK2 kinase domain and overactive JAK/STAT [However, as is the case for many targeted therapies, treatment failure can occur in the setting of intrinsic resistance to JAK or TYK2 inhibitory drugs, or development of resistance to these drugs over time ,157. Forpathways ,163,164.K2 V617F . SimilarMYB-TYK2 cells with acquired cerdulatinib resistance, histone deacetylase inhibitor (HDACi) therapy proved efficacious in blocking proliferation [Development of combination therapies is one approach to combat resistance. Recent studies have evaluated several combination therapies in attempts to improve overall efficacy or overcome resistance to TYKinibs/JAKinibs or other cancer drugs ,157,164.feration ,166. Indferation . Ruxolitferation . In addiferation . Similarferation . TYK2 mutations or expression levels may help determine patient prognosis. In addition, with the development of multiple pharmacologic inhibitors of TYK2 and JAKs, those tumors that are TYK2-positive may be viable candidates for TYKinib/JAKinib targeted therapies. Future pre-clinical studies should be geared at evaluating therapeutic interventions with TYK2 inhibitors as single agents, or in combination therapy to pave the way for biomarker-driven clinical trials.Personalized, or precision, medicine has increasingly become a valuable tool in clinical diagnostics to evaluate potential therapeutic options for individual cancer patients . Based o


Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC ; however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of  Driven by the recent development of next- and third-generation sequencing technologies, myriads of single-cell amplified genomes and metagenome-assembled genomes have been retrieved from diverse environments using multi-omics approaches . These iCandidatus Pelagibacter”) (Candidatus Puniceispirillum”)  has been successfully applied to oligotrophic marine ecosystems by inoculating microbial cells into a physically separated microwell with natural seawater-based media . Using Hbacter”) , SAR 116rillum”) , OM43 (Grillum”) , SAR92 (rillum”) , OM60 (Crillum”) , SUP05 (rillum”)  and otheAlphaproteobacteria, Betaproteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Chloroflexi, and Verrucomicrobia , a freshwater methylotrophic Betaproteobacteria (Candidatus Fonsibacter”), a freshwater sister group of the marine SAR11 clade , the predominant actinobacterial lineage found in most freshwater lakes  and water overturn begins in early winter and lasts until the surface water warms again in spring. Therefore, Lake Soyang is classified as a typical warm monomictic lake. In Korea, heavy rainfall occurs during the monsoon season (July–August) and flows into the reservoir’s metalimnion . Therefoin situ with a YSI 556 MPS Multiprobe System . Concentrations of ammonium, nitrite, nitrate, phosphate, silicate, total dissolved nitrogen, and dissolved organic carbon were determined by the National Instrumentation Center for Environmental Management (NICEM), Korea, following the Standard Methods for Examination of Water and Wastewater guidelines .The temperature, pH, dissolved oxygen, conductivity, and salinity of each water sample were measured idelines . After p5 cells/ml were recorded as growth-positive, after which 400 μl of these growth-positive cultures were cryopreserved in a glycerol suspension  at −80°C. Of the remaining 500 μl of culture, 100 μl was transferred to a microcentrifuge tube to serve as PCR template, and 10 μl was spotted onto Reasoner’s 2A (R2A)   and 1/10Growth-positive culture samples were freeze-thawed three times in microcentrifuge tubes and used directly as PCR templates using the 27F-B and 1492R universal bacterial primers  and a thThe colony-forming activity of liquid HTC cultures was tested by spotting 10 μl of culture samples on R2A and 1/10R2A plates . The inoculated plates were then incubated at 15°C for 1 month and visually inspected to assess colony formation. Using the colonies formed on the agar plates, 16S rRNA gene sequences were determined via the bead-beating method and the sequences were compared with those of the initial liquid-cultured HTC isolates. Once the identity of the colonies was confirmed, additional sequences were obtained using the 518F primer (5′-CCA GCA GCC GCG GTA ATA CG-3′) and assembled with the sequences obtained with the 800R primer. Colonies were also cryopreserved as 10% glycerol suspensions at −80°C.For the HTC isolates that did not form colonies on R2A or 1/10R2A, HTC-isolated glycerol stocks were used to test whether cryopreserved isolates could resuscitate during the freeze-thawing process. At least one representative strain from each phylogenetic group  represented in the maximum-likelihood trees was selected and retrieved from the glycerol stocks. Cryopreserved stocks were thawed at room temperature, after which 200 μl of the stocks were inoculated into 20 ml of fresh media with the same composition as the media from the initial HTC experiments. Cellular growth during incubation at 15°C was monitored via flow cytometry every week and isolates with a 10-fold increase in cell numbers relative to their initial cell densities were considered “reviving-positive.” Bacterial strains whose growth was not observed after 4 weeks of incubation or exhibited a less than 10-fold increase were considered “reviving-negative.” Finally, upon confirming that the 16S rRNA gene sequences of the revived cultures matched those of the initial HTC isolates, additional sequences were obtained using the 518F primer as described above.To analyze the bacterial community diversity in Lake Soyang, 1 L of eight lake water samples was filtered through a 0.2 μm-pore-size 47 mm polyethersulfone membrane filter  after prefiltration with a 3.0 μm-pore-size membrane filter. After cell lysis by lysozyme treatment on membrane filters, DNA was extracted using the DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. The V1-V3 region of the 16S rRNA genes was amplified using fusion primers, whose design was based on that of the 27F and 519R universal primers. Pyrosequencing was performed by ChunLab, Inc.  using a Roche 454 GS Junior sequencer.n = 6,266 sequences in this study). Operational taxonomic unit (OTU) clustering was conducted using an average-neighbor algorithm at a 98% similarity cutoff, then normalized for further statistical analysis. Distance calculation by the Bray-Curtis method, principal coordinate analysis (PCoA), and hierarchical clustering using the unweighted pair-group method with arithmetic means (UPGMA) was conducted using the vegan package in R version 3.3.2  was detected in 14.0%  of the inoculated wells , summer (July 2014), autumn (October 2014), and winter (January 2015). The studied lake exhibited a thermocline in spring and summer , and phyed wells . The culed wells , showed ed wells . It is ned wells .5 cells/ml elucidated the presence of 605 putatively pure cultures and 145 mixed-cultures, and therefore only the axenic cultures were used for further phylogenetic analyses , Bacteroidetes (36 strains), and Verrucomicrobia (5 strains) and the classes Alphaproteobacteria (62 strains), Betaproteobacteria (449 strains), Gammaproteobacteria (3 strains), and Deltaproteobacteria (1 strain). Maximum-likelihood trees generated using the sequences from these 605 pure cultures and their relatives indicated that the HTC isolates obtained in this study were affiliated with diverse phylogenetic clades comprising different lineages, tribes, or genera , Betaproteobacteria (449 strains), Gammaproteobacteria (3 strains), and Deltaproteobacteria (1 strain). The HTC isolates belonging to the Alphaproteobacteria were affiliated with 22 phylogenetic groups comprising five orders  (Hirschia were found exclusively in summer), these trends cannot be generalized due to the small number of isolates for each phylogenetic group. Further, although many isolates of Alphaproteobacteria belonged to well-defined genera , 22 isolates belonged to as-yet-uncultured lineages such as A0839, Alpha1, FukuN57, MNG7, Wr0007, and GOBB3-C201. In the SILVA database, the A0839 lineage currently contains > 2,300 16S rRNA gene sequences of uncultured bacteria retrieved from diverse aquatic or terrestrial habitats including an Antarctic lake (Candidatus Fonsibacter”), has been reportedly isolated from brackish water   were assigned to the phylum nadales) , all of nadales) . Among ttic lake  and a metic lake . Given talinity) , suggestGammaproteobacteria and one strain of Deltaproteobacteria were isolated throughout the study , LimD (36), a new Limnohabitans group (Lim 2) represented by IMCC26036 (52) (Rhodoferax_Albidiferax (53), PnecA (20), PnecB2 (24), PnecC (39), and a new Polynucleobacter group (Pnec1) represented by IMCC26046 (13)  of the order Methylophilales. These groups were all found to belong to four well-known freshwater bacterial genera: Limnohabitans, Polynucleobacter, Rhodoferax (including Albidiferax), and “Candidatus Methylopumilus” (LD28 freshwater group). In addition to these groups, some HTC isolates were identified as members of the MWH-UniP1, GKS98, and TRA3-20 groups that currently do not have any cultured representatives. At least one strain from the three groups formed colonies, which will likely prove to be an important resource for future taxonomic and genomic studies. In total, 68 betaproteobacterial isolates exhibited colony-forming capacities, and most of them (50 isolates) were identified to be members of the genus Polynucleobacter (Within the phylum orldwide . In our ntatives . Most be036 (52) , Rhodofe046 (13)  of the oeobacter .Betaproteobacteria belonged to groups related to the genus Limnohabitans  of habitans , 2. The osystems  and is cosystems . Based oproposed . Upon aps herein . Concreton group , the inahabitans .Polynucleobacter was the second largest group within the HTC isolates (96 isolates) and was further distributed into the lineages PnecA (20 isolates), PnecB2 (24), PnecC (39), and a newly identified lineage named Pnec1 (13)  . Pnec1 wec1 (13) . The genal group , 2016a,  species . Eleven perature . LikewisMethylophilaceae. This family is one of the key methylotrophic bacterial groups in freshwater and marine habitats , in addition to the LD28 freshwater group (“Candidatus Methylopumilus planktonicus”) (Candidatus Methylopumilus planktonicus) (Methylotenera (3)  , OM43 maonicus”) , and PRDtonicus) , the memtonicus) . These Hnera (3) . Interese Zurich . The 16Se Zurich . Among te strain , numerouActinobacteria is considered the most abundant bacterial group in freshwater lakes, often accounting for > 50% of limnetic bacterioplankton , Acidimicrobiales (19 strains), and Gaiellales (25 strains) (The phylum plankton . In our strains) . HoweverFrankiales belonged to the acI lineage, the predominant freshwater actinobacterial group  and an acIV-D clade, which will be eventually renamed    based onGaiellales, a total of 25 HTC isolates were affiliated with two new groups defined in this study for which cultured representatives have not yet been reported. These two groups were named “unidentified Gaiellales 1” (3 isolates) represented by strain IMCC29153 and “unidentified Gaiellales 2” (22 isolates) represented by strain IMCC29033   . Moreovec matter .Verrucomicrobia: OPB35_soil group , Opitutae (1 strain), and Prosthecobacter (1 strain)  . Howeveror xylan . TherefoAlphaproteobacteria); PnecB2, Lim1, and LimB (Betaproteobacteria); acIV, uncultured Gaiellales group 2, and “Candidatus Limnosphaera” (Actinobacteria); and OPB35_soil group (Verrucomicrobia). Among these, two strains of acIV, four strains of Limnohabitans, and strain IMCC26040 of the OPB35_soil group were selected for growth curve determination. The seven strains reached their maximum cell densities at 10-20 days of incubation in axenic conditions, exhibiting fastidious but stable growth characteristics  OTUs were obtained from eight freshwater samples used as inocula during the HTC trials . The frebacteria .Proteobacteria, Actinobacteria, Bacteroidetes, Verrucomicrobia, Acidobacteria, Planctomycetes, Chloroflexi, and Cyanobacteria) accounted for more than 1.0% of the total reads. The phyla Proteobacteria and Actinobacteria were always dominant in all samples, accounting for 35.4−67.1 and 20.3−40.8% in 1 m depth samples, and 26.7−37.8 and 29.8−42.0% in 50 m depth samples, followed by Bacteroidetes (average 8.5%), Verrucomicrobia (5.5%), Acidobacteria (4.1%), Planctomycetes (2.7%), Chloroflexi (2.1%), and Cyanobacteria (1.9%)  and Betaproteobacteria (17.5%) were the predominant classes in all samples, whereas Gammaproteobacteria (3.7%) and Deltaproteobacteria (1.0%) were only present in far lower proportions. However, their abundance varied with season and water depth.Among the 29 phyla identified, eight phyla (a (1.9%) . Within Alphaproteobacteria in the summer surface  of Lake Soyang exhibited more dramatic seasonal variations than those of the hypolimnetic layer (50 m) . Therefo surface . These vProteobacteria, Actinobacteria, Bacteroidetes, and Verrucomicrobia) could be cultivated. Furthermore, members of the class Betaproteobacteria were abundantly found in both the culture-independent analyses  and the HTC experiment , suggesting that the HTC culture conditions favored the growth of Betaproteobacteria. Similarly, the Limnohabitans, Polynucleobacter, and “Candidatus Methylopumilus” genera, which accounted for high proportions of the HTC isolates, were also abundantly present in the bacterial community of Lake Soyang , which was primarily represented by the acI lineage, appeared to be consistently abundant regardless of season or depth , the LD28 freshwater group (4.6%), and LimB (2.5%), confirming that these groups were successfully cultivated using the HTC method employed herein.To determine the proportion of the total bacterial community represented by the HTC isolates, the 605 sequences of the HTC isolates were queried via BLASTn analysis against eight pyrosequencing data sets retrieved from Lake Soyang. These analyses indicated that 26.7 and 24.5% of the pyrosequencing reads corresponded to the 16S rRNA gene sequences of the HTC isolates based on 97.0 and 98.7% sequence similarity thresholds, respectively . The phyPolynucleobacter, and Limnohabitans lineages, whose pyrosequencing tags were largely dominant. The HTC method using media based on ambient lake water allowed for the cultivation of these dominant bacterioplankton groups. However, some of the major lineages and tribes including LD12, acI-A6, -A7, -B4, and -C2 could not be cultivated with this approach. Considering the small number of HTC isolates belonging to the major bacterial lineages observed in this study, the medium composition and culture conditions used herein may have not favored some of the aforementioned lineages. Therefore, to obtain stably growing axenic cultures of fastidious microorganisms, future studies must focus on the development of cultivation strategies based on genomic and ecological data. One remarkable example of such an approach is the recent successful cultivation of the genome-streamlined acI lineage by supplementing its growth media with chemical compounds required for their growth, including reduced sulfurs, amino acids, vitamins, and catalase (Overall, our results indicated that the bacterial community composition of Lake Soyang was similar to that of many lacustrine environments worldwide . Particucatalase , 2020. TAcidobacteria could not be cultured using HTC. Therefore, additional studies are required to optimize the HTC growth conditions of these freshwater lineages by integrating the ever-increasing wealth of related genomic and ecological data.In this study, we conducted HTC experiments and tag-encoded pyrosequencing analyses using water samples collected from the epilimnion and hypolimnion of Lake Soyang over four seasons. This resulted in the isolation of more than 600 bacterial strains and enabled the characterization of the bacterial community structure of Lake Soyang, a freshwater reservoir. The Lake Soyang bacterial community was comprised of phylogenetically diverse bacterioplankton groups, most of which belonged to cosmopolitan freshwater bacterioplankton lineages, but whose populations varied dramatically in a season- and depth-dependent fashion. The isolates cultured via HTC were assigned to many predominant and previously uncultivated or underrepresented phylogenetic groups, including multiple tribes of the acI, acIV, LD28, FukuN57, MNG9, and TRA3-20 lineages. Nevertheless, despite the successful cultivation of previously uncultivated freshwater bacterioplankton groups, many abundant bacterial lineages such as LD12 and MW884569–MW885173. The accession numbers for each sample are also listed in SRR14278392–SRR1427839.The 16S rRNA gene sequences of the HTC isolates were deposited in the GenBank/EMBL/DDBJ databases under accession numbers SK and MI performed the wet experiments. SK and IK performed bioinformatic data analyses. IK and J-CC designed and directed the study. SK, MI, IK, and J-CC analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure. Phosphodiesterases (PDEs) are the enzymes that degrade both cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) to nucleoside phosphates . PDEs are categorized in 11 families with subfamilies and splice variants ,3. All PIn this review, we focus on the role of PDEs in regulating cardiac contractility . After c2+ channels (LTCC) and influx of extracellular Ca2+, which in turn triggers the opening of ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR) that release Ca2+ into the cytosol. Intracellular Ca2+ binds to TnC, leading to a conformational change and binding to TnI; this in turn moves its inhibitory domain away from actin and promotes the displacement of tropomyosin, leaving the actin surface free to bind the myosin heads. Together, these conformational changes lead to formation of cross-bridges along the myosin and actin myofilaments. When the cross-bridges are formed, ATP binds to myosin. Its hydrolysis brings the myosin heads to produce the power stroke, and the myofilaments will slide past each other. This triggers muscle shortening and ultimately contraction of the heart, called systole. Following contraction, cytosolic Ca2+ is efficiently removed and the cytosolic Ca2+ levels drop, due primarily to the activity of the sarcoplasmatic reticulum Ca2+-ATPase (SERCA) and the sarcolemmal Na+/Ca2+ exchanger, but also the sarcolemmal ATPase and the mitochondrial Ca2+ uniporter. As calcium levels drop, calcium bound to TnC is released, and TnI and tropomyosin are returned to a closed state that prevents actin from interacting with myosin. Thus, relaxation of the heart (diastole) can occur [2+ removal as well as the contractile proteins themselves are regulated by, e.g., cAMP and cGMP, mainly by phosphorylation of proteins involved in Ca2+ handling, such as LTCC, RyR, and the SERCA-inhibiting protein phospholamban (PLB), and sarcomeric proteins, such as TnI, myosin binding protein C (MyBP-C), and titin. Effects that enhance contractility, i.e., the ability of the heart to contract, are called (positive) inotropic effects, whereas effects that enhance cardiac relaxation are called (positive) lusitropic effects.Cardiomyocytes are the key players in cardiac contraction and relaxation. They contain contractile structures composed of the myofilaments, myosin and actin, organized in sarcomeric units. Along the myofilaments, actin monomers are localized with tropomyosin and the complex of troponin T, troponin I, and troponin C  . Electrian occur . The effs. Gi-coupled GPCRs, on the other hand, activate the inhibitory G protein Gi that inhibits AC activity. Cyclic AMP activates several effector proteins, such as protein kinase A (PKA), exchange proteins directly activated by cAMP (Epac1 and Epac2), cyclic nucleotide-gated ion channels (CNGCs), and Popeye-domain-containing proteins, which regulate a vast array of downstream processes. PKA, as the most important cAMP-activated effector in regulation of contractility, is spatially localized and regulated by a family of scaffolding proteins termed A Kinase Anchoring Proteins (AKAPs). AKAPs anchor PKA to its target and the complexes often include PDEs responsible for regulating local cAMP levels, and phosphatases to tightly regulate PKA-mediated phosphorylation [Cyclic AMP is a second messenger synthetized by adenylyl cyclase (AC) that can be activated in response to stimulation of G-protein-coupled receptors (GPCRs) coupled to the AC-stimulating G protein Grylation ,10,11,123-AR [Cyclic GMP is synthesized by soluble guanylyl cyclase (sGC), activated by nitric oxide (NO), which is in turn synthesized by nitric oxide synthases (NOS); particulate guanylyl cyclases (GCs) can also synthesize cGMP when activated by the natriuretic peptides (NP) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) . Increas3-AR . In orde3-AR ,16.2+ into the SR. Phosphorylation of TnI, on the other hand, reduces the sensitivity of the myofilaments for binding Ca2+, thus allowing faster Ca2+ dissociation. Together, this results in a faster relaxation (lusitropic response) [2+ is not limiting, as under cAMP stimulation, PLB phosphorylation will also contribute to a positive inotropic effect through increasing SR Ca2+ content. PKG can also phosphorylate PLB and TnI, causing a LR similar to PKA phosphorylation [2+ availability due to phosphorylation of PLB and LTCC, combined with TnI phosphorylation, PKG activation is mostly associated with a negative inotropic response (NIR) [2+ may be limiting due to lack of LTCC stimulation, and PLB phosphorylation will mainly contribute to a NIR [Cyclic AMP and cGMP activation of PKA and PKG, respectively, modulates contractility through phosphorylation of several proteins. PKA phosphorylation of LTCC and RyR increases their open probability and therefore increases cytosolic calcium levels. Together, this generates a positive inotropic response (PIR). Cyclic AMP also mediates a lusitropic response (LR) through PKA phosphorylation of PLB and TnI. Phosphorylation of PLB removes its inhibitory tone on the SERCA pump, which results in increased pumping of Caesponse) . In siturylation ,22,23,24se (NIR) ,27,28,29se (NIR) ,31. Thus1 did not, despite both increasing cAMP [1 was found only in the soluble fraction [Intracellular signaling mediated by cyclic nucleotides (CNs) is organized in spatial-temporal nanodomains defined by localization and activities of receptors, adenylyl or guanylyl cyclases, protein kinases and PDEs, and this is defined as compartmentation. The first evidence for compartmentation of second messenger signaling in the heart was published in the 1970s and early 1980s for cAMP, where it was observed that isoproterenol (Iso) stimulation of β-ARs increased contractility, while stimulation with the prostaglandin PgEing cAMP ,33,34. Ifraction ,36.1-adrenergic receptor (β1-AR) is the main β-AR in cardiomyocytes. Stimulation of these receptors activates Gs that activates ACs, of which AC5 and AC6 are the main isoforms expressed in the heart [2-ARs represent a smaller portion of ARs in the heart and also activate Gs (as β1-AR). Whereas β2-AR stimulation can trigger a PIR, its ability to trigger a LR was disputed [2-ARs to activate both Gs and Gi but may also be related to the receptor localization. In this regard, β2-ARs are found in the T-tubules, and thereby specifically regulate LTCC current [1-AR stimulation produces cAMP both in the T-tubules and at the cell crest [2-AR signaling seems very restricted, while the β1-AR-stimulated cAMP can diffuse more throughout the cell and reach multiple targets. Similar to β-AR signaling, the serotonin 5-HT4 receptors can also give an increase in cAMP and a PIR, at least in failing hearts [The GPCR βhe heart . Increashe heart . β2-ARs disputed , althougdisputed . The red current , while βll crest ,46,47,48g hearts ,51,52,53Cyclic AMP compartments relevant for contractility are regulated by several PDEs. Amongst the cAMP-selective PDEs in the heart, PDE4 is the main PDE, whereas little is known about PDE8.m (1.2–10 µM). It comprises a family of 20 isoforms encoded by PDE4A, B, C, and D genes, of which all except PDE4C are found in the heart [1/β2-AR mediated contractility, while inhibition of PDE3 did. However, also in human failing myocardium, whereas PDE4 inhibition alone had no effect, a role of PDE4 inhibition in enhancement of a 5-HT4-mediated positive inotropic response to serotonin was revealed in the presence of PDE3 inhibition [PDE4 is a cAMP-selective PDE with a low Km .2–10 µM.he heart . The N-the heart ,58,59,60he heart . Howeverhe heart ,62,63,64he heart  or carvehe heart , showed hibition . On a si1-AR-mediated PIR and LR, has been reported in both atria and ventricle [2-AR-mediated contractility, there are regional differences in the heart, where PDE4 regulates contractility in the base but not the apex of the ventricle. This could be attributed to several factors, including more regular T-tubular organization and higher PDE4 activity at the base of the heart [2-AR-stimulated cAMP near PLB, whereas PDE2 and PDE3 did [1-AR is regulated by PDE4 near PLB. One study has also reported localization of PDE4 near PLB in human failing myocardium [In rodents, PDE4 is the main PDE responsible for hydrolysis of cAMP, when measuring cAMP levels, and together with PDE3 regulates β-AR-stimulated contractility ,68,69,70entricle ,71,72. Fhe heart . In one he heart . In anotPDE3 did . Taken tocardium .2+ leak and in heart failure (HF) reduced local PDE4 gives increased phosphorylation of RyR and greater Ca2+ efflux from the SR [2+ by being part of a complex with SERCA, PKA, and PLB [2+ entrance in the cell when phosphorylated by PKA [PDE4D3 forms a signalosome together with PKA and RyR regulating the SR-Cam the SR . Moreove and PLB . PDE4B id by PKA ,79. Reced by PKA . This cod by PKA .1-AR-, β2-AR-, or 5-HT4-mediated contractility was not enhanced by inhibiting PDE4 alone, despite increased levels of cAMP. Rather, simultaneous inhibition of both PDE3 and PDE4 was needed to obtain an increased contractility, both in normal and failing hearts [4 receptor stimulation, inhibition of both PDE3 and PDE4 significantly increased cAMP levels in porcine left atrium [In some reports on human, porcine, and rodent cardiac ventricle, βg hearts ,82,83,84t atrium  and in ht atrium . Togethet atrium .−/− cardiomyocytes have larger Ca2+ transients, increased LTCC current, and increased cardiomyocyte contractility upon β-AR activation, but also larger Ca2+ spark frequency in the absence of receptor activation [1-AR caused a widespread cAMP signal, whereas β2-AR caused a much more localized cAMP signal [2-AR cAMP signal more similar to that of β1-AR. However, the difference was insensitive to PDE inhibition by IBMX, which at the time would indicate that PDEs were not involved. However, an intriguing possibility is that PDE8 could be involved in the difference, and that PDE8 inhibition, maybe in addition to IBMX, would make the β2-AR signal more like the β1-AR signal.PDE8 is a cAMP-selective phosphodiesterase and has two isoforms: PDE8A and PDE8B . The regtivation . This suP signal . If thisOne of the first indications of cGMP compartmentation was seen in the early 1990s when cGMP from NO was found to be in both the soluble and the particulate fraction, while cGMP from the pGC was present only in the particulate fraction . In rat The PDEs that selectively hydrolyze cGMP in the heart are PDE5 and PDE9.PDE5 selectively degrades cGMP and has three isoforms (PDE5A1–3) expressed in human hearts . The reg2-AR-mediated contractility after PDE5 inhibition [3-AR activation, at caveolae-enriched membrane fractions in T-tubules. This cGMP pool was then responsible for a decrease in cAMP levels through PDE2 activation [3-ARs were found to elicit a NIR and positive LR via eNOS and cGMP [Inhibiting PDE5 does not affect basal contractility. However, when activating all three β-ARs, PDE5 inhibition (by sildenafil) reduced contractility in normal mouse heart involving increase in NOS3-NO-sGC-originated cGMP ,103, as hibition . In additivation . These dand cGMP ,110. In and cGMP .m for cGMP (0.70–0.17 µM) and it was recently discovered to be expressed in the heart, localized to the SR , and regulating cGMP generated after GC-A stimulation, but not from stimulating sGC [PDE9A is the PDE with the lowest Kting sGC ,105. Morting sGC . We are Although the cAMP- and cGMP-pathways modulate contractility differently, as described above, increased concentrations of cGMP can also modulate cAMP levels, by affecting the activity of certain PDEs. Cyclic GMP binds to a GAF domain on PDE2 to increase the enzymatic activity, and thereby reducing cAMP responses, whereas at PDE3 cGMP is hydrolyzed slowly and this inhibits cAMP degradation, increasing cAMP-mediated responses. Thereby, PDEs that can hydrolyze both cAMP and cGMP are responsible for so-called cross-talk between the two signaling pathways. The compartments relevant in contractility that involve such cross-talk are not easy to differentiate from the specific cAMP/cGMP compartments described above. Further investigation with focus on local cyclic nucleotide fluctuations and on how subcellular compartmentation occurs, will help to resolve this.The dual-substrate PDEs also have important roles towards the two single pathways, and in the next section, we describe how PDE1, PDE2, and PDE3 contribute to modulate contractility by regulating either cAMP or cGMP separately, but also how they modulate contractility by creating cGMP/cAMP cross-talk.2+/calmodulin (CaM)-dependent enzymes and their regulatory domain contains two CaM binding sites, two phosphorylation sites, and an inhibitory region, which retains the enzyme in its inactive configuration when the calcium levels are low [2+/CaM and therefore its activation [m than for cAMP, while PDE1C has equal affinity [The PDE1 family includes the isozymes PDE1A, PDE1B, and PDE1C . These a are low ,114. CaMtivation ,116. PDEaffinity . Among taffinity ,118,119.affinity .Most studies report data on the functional role of PDE1 in rodent hearts and do not investigate its specific effect on contractility. Recently, the group of David Kass has shown the role of PDE1C in rabbit and dog hearts, which, similar to human hearts, express predominantly this isoform and show positive inotropic and chronotropic effects after inhibiting PDE1. These effects are due to PDE1C activity over a soluble AC-cAMP nanodomain that regulates LTCC current via PKA, but does not involve modulation of SERCA current, phosphorylation of PLB, TnI, and myosin binding protein C, like in the β-AR-mediated contractility ,121.max and high Km for both cAMP and cGMP [PDE2 is a dual substrate phosphodiesterase with similar high Vand cGMP ,123. Theand cGMP ,125,126.and cGMP  and in cand cGMP .The regulatory region contains two GAF domains, A and B . Cyclic In rat and mouse cardiomyocytes PDE2 contributes to a low fraction of the total cellular cAMP-PDE activity ,110, whiTotal cGMP levels seem to be mainly controlled by PDE2 both in normal  and fail1- and β2-AR activation [3-ARs trigger NO release and consequent cGMP augmentation and that these pools of cGMP are responsible for PDE2 activation and thereafter reduction in β1- and/or β2-AR-stimulated cAMP levels and PIR [3-ARs in the caveolae and the related cGMP pools in the T-tubules, which are locally regulated mostly by PDE2 and PDE5 [Considering that PDE2 is a dual substrate PDE, and that cGMP can bind to the PDE2-GAF domain and increase its enzymatic activity, the role of PDE2 in cGMP/cAMP cross-talk has been investigated. Specifically, cGMP-increase after sGC stimulation reduces LTCC current through activation of PDE2 and this is mediated by a decrease in cAMP and PKA phosphorylation of LTCC ,137,138.tivation , and suctivation . Mongill and PIR . Supportand PDE5 , and stiand PDE5 .1- and β2-AR compartments, revealing an ANP-induced increase of the positive inotropic effect of β-adrenergic stimulation in a mild hypertrophic model through PDE3 inhibition [1-AR microdomain and increased around the β2-AR microdomain, therefore, ANP-stimulated cGMP will switch from reducing cAMP levels via PDE2 activation  to increase cAMP levels via PDE3 inhibition (in a disease model) relatively to the β1-AR microdomain [CNP can also, through cGMP-mediated activation of PDE2, decrease cAMP levels and thereby decrease β-AR-stimulated LTCC current ,140. Evehibition . In thisrodomain .m cAMP 0.16 µM; Km cGMP 0.09 µM), while the Vmax is >10-fold higher for cAMP. Hence, when cGMP binds, it is hydrolyzed slowly and this inhibits cAMP degradation [2+ re-uptake in the SR [PDE3 has two isoforms, PDE3A and PDE3B, both expressed in the heart ,143,144.radation . The N-tradation . The latradation ,148,149.n the SR ,151.4 receptor-mediated PIR and LR [PDE3 is responsible for hydrolyzing global cellular cAMP levels in hearts from normal and HF animals, to a lower extent than PDE4, which is the main cAMP-PDE in rodents ,68,74. AR and LR ,52,98 anR and LR ,155.2-AR signaling in caveolin-enriched compartments such as T-tubules [2-AR-cAMP at the plasma membrane was reduced, with no changes in global expression, but with clear reduction in the caveolin-rich fractions [4-receptor-induced cAMP levels, calcium, PIR and LR, compared to inhibiting either PDE alone [Using targeted FRET-based biosensors, PDE3 subcellular activity was found to prevail around cAMP nanodomains defined by a PKA RI-targeted biosensor  and regu-tubules . Howeverractions . Similarractions . TherefoDE alone ,84,156.With regard to cGMP, PDE3 regulates global cellular cGMP levels and cytosolic cGMP measured by a non-targeted biosensor  and cont1-AR and β2-AR in normal and failing rat left ventricle [4 receptor-induced PIR was also shown to be increased by CNP-induced PDE3 inhibition in both failing rat left ventricle and in porcine left atrium [4-mediated PIR (through PDE3 inhibition), but also reduced β1-mediated IR [1-mediated cAMP and IR by NO/sGC has been shown by others as discussed above under “PDE2 and cGMP/cAMP cross-talk relevant for contractility” [1-AR and 5-HT4 receptors may indicate different compartmentation of PDEs in their signaling pathways. As also addressed above, ANP potentiated the β-AR-stimulated PIR, presumably through PDE3 inhibition in a mild hypertrophic model, but not in Sham [1-AR-dependent contractility, neither in a chronic HF model nor in sham-operated animals [The natriuretic peptide CNP can modulate contractility by affecting cAMP signaling through cGMP-mediated inhibition of PDE3, causing a PIR mediated by βentricle ,62,98. 5t atrium ,81. In Hiated IR . The redctility” ,134,139. in Sham . However animals ,98.PDE10 is a dual PDE discovered in 1999 ,158,159 In HF the heart fails to provide adequate blood flow/pressure to meet the body’s demands and this is associated with alterations in the cardiac structure, function, rhythm, and conduction. In HF, the body responds by activating compensatory mechanisms, such as salt and water retention, increase in noradrenaline and adrenaline (sympathetic activation), and increase in several hormones, such as angiotensin II, aldosterone, endothelin, and natriuretic peptides. Some of these changes will in the short term improve cardiac contractility and cardiac output, but in the long term some of these will worsen the prognosis ,161. Of Despite the fact that cAMP and cGMP have many similarities in the signaling pathway and have similar protein targets, they can mediate different and sometimes opposite effects on contractility in the heart, as described above. In HF there is increased adrenergic activity stimulating β-ARs and increased natriuretic peptides. Long-term stimulation of β-AR is detrimental in chronic heart failure and β-blockers are a cornerstone in HF management ,163. IncPDE2 expression increases in HF ,180. HowIn heart failure, alteration in PDE3 expression is not that clear. Some studies show a reduction in both humans and animal models ,183,184,In HF expression of both PDE4A and PDE4B, as well as PDE4 activity, is reduced and accompanied by heart dysfunction ,184,186.In a recent study, PDE4B overexpression both decreased contractility and lusitropic responses through reduced phosphorylation of PLB, TnI, and MyBPC . In addiPDE5 activity is enhanced in HF, both in humans and in animal models ,208,209 The expression of PDE9 in the heart is low, but it is upregulated in human left ventricular biopsies from HFpEF and aortic stenosis patients and in animals with HFrEF . Inhibitm. However, inhibition of PDE2 increased cell shortening at basal conditions [For many years, studies on the intracellular signaling modulating contractility have focused on measuring global cAMP levels in relation to a specific functional response. However, we now understand more of the organization of the compartmented signaling networks relaying responses, from GPCR activation to cAMP signaling in subcellular nanodomains . Conseqnditions . Similarnditions , but whenditions . Targetinditions . These oLocalization of signaling in subcellular compartments could also explain the discrepancy between global cAMP concentrations at basal conditions, which are around 1 µM, and PKA affinity for cAMP, which is ~100 nM ,224,225.Since PDE inhibitors were first used for treatment of HF, many advances have been made in understanding the role of PDEs in cardiac contractility. However, there are some ongoing challenges to consider. For example, a large number of studies are conducted using animal models where PDE activity and expression can be different from those in humans, and thus caution is needed when interpreting the data. As we appreciate more the importance of signaling compartmentation, localization of specific PDEs is also of relevance in modulation of contractility.Real-time measurements of cAMP and cGMP using targeted biosensors has been pivotal in understanding how PDEs contribute to mediate contractility at the subcellular level and helped to uncover signaling compartmentation. Yet, there are still some questions and challenges remaining: how do PDEs efficiently degrade cAMP/cGMP to protect protein kinase from unwanted cellular activation? How does stimulation of a specific receptor trigger the activation of certain downstream targets and in some compartments rather than others? Moreover, how are the compartments involving cGMP/cAMP cross-talk organized vs. the single cAMP/cGMP compartments?Using targeted biosensors combined with in silico modeling will help to understand the compartments in a cell, which modulators are involved, the organization of cAMP/cGMP local nanodomains and their size, and how these are affected in cardiac dysfunction.Heart failure has been associated with changes in either expression or localization of PDEs. In addition, there is evidence that some PDEs can behave in a redundant manner. How one PDE may compensate for changes in the activity of another PDE during signaling modulation or whether this redundancy changes during HF, remains to be fully elucidated.


Xanthomonas axonopodis pv. phaseoli (Xap), is one of the most destructive diseases of common bean (Phaseolus vulgaris L.). The tepary bean genotype PI 319443 displays high resistance to Xap, and the common bean genotypes HR45 and Bilu display high resistance and susceptibility to Xap, respectively. To identify candidate genes related to Xap resistance, transcriptomic analysis was performed to compare gene expression levels with Xap inoculation at 0, 24, and 48 h post inoculation (hpi) among the three genotypes. A total of 1,146,009,876 high-quality clean reads were obtained. Differentially expressed gene (DEG) analysis showed that 1,688 DEGs responded to pathogen infection in the three genotypes. Weighted gene coexpression network analysis (WGCNA) was also performed to identify three modules highly correlated with Xap resistance, in which 334 DEGs were likely involved in Xap resistance. By combining differential expression analysis and WGCNA, 139 DEGs were identified as core resistance-responsive genes, including 18 genes encoding resistance (R) proteins, 19 genes belonging to transcription factor families, 63 genes encoding proteins with oxidoreductase activity, and 33 plant hormone signal transduction-related genes, which play important roles in the resistance to pathogen infection. The expression patterns of 20 DEGs were determined by quantitative real-time PCR (qRT-PCR) and confirmed the reliability of the RNA-seq results.Common bean blight (CBB), primarily caused by  Phaseolus vulgaris L.), a legume that originated in the Americas, belongs to the Fabaceae family and is one of the most important edible legumes in the world  and Xanthomonas fuscans subsp. fuscans (Xff). The pathogenic bacterium Xff can be easily identified as it can produce a brown pigment in milk Tween (MT) agar medium  and runner bean (Phaseolus coccineus) (CBB is a seed-borne disease caused by r medium . Althougin China . The priccineus) . Under hccineus) . It has ccineus) . Crop roP. coccineus and P. acutifolius, have a higher level of resistance, using interspecific crosses is a useful way to breed resistant lines. The breeding lines HR45 and HR67, which have BC420 and SU91 QTLs with pyramided resistance, were derived from XAN159, which was developed from an interspecific cross between ICI Piajo and P. acutifolius PI 319443  for CBB resistance have been mapped, and these are primarily located on chromosomes 2, 3, 6, 7, 8, and 11 . HoweverI 319443 . HoweverXanthomonas phaseoli pv. phaseoli, and resistance was found to be associated with the salicylic acid pathway and sugar metabolism; moreover, the genes involved were modified , functional gene mining, and identifying key components of disease resistance pathways in plants . Based omodified . In thisThe resistant genotypes HR45 and PI 319443 and the susceptible genotype Bilu were used in the present study . HR45 waXap strain XS2, isolated and preserved by our laboratory, is the most widespread and harmful pathogen in China. To obtain fresh bacterial cultures, suspensions were grown for 48 h in nutrient broth at 28°C at 220 rpm. MT medium was prepared according to the International Seed Testing Association (ISTA) method (The ) method .8 colony-forming units (CFU)/mL in sterile distilled water. Eight days after planting, Xap inoculation was carried out when the primary leaves were fully expanded, and the relative humidity and temperature were increased to 95% and 28°C/26°C (light/dark) to provide the appropriate conditions for inoculation. Pathogen inoculation was conducted by injection as follows: a 200 μL pipette tip dipped in bacterial solution was used to pierce the leaf at three points on the right side at a distance of approximately 0.5 cm from the central main vein, allowing the pathogenic bacteria to enter the leaf through the wound to infect the host tissue; the left side of the leaf was inoculated with sterile water as a control. After the completion of inoculation, the sample was incubated at 28°C with 95% humidity. The severity of disease in each inoculated leaf was investigated and recorded with reference to the rating criteria  was used to specifically amplify XS2 DNA . The DNA was extracted, and its concentration was adjusted to 50 ng/μL. qRT-PCR was used to calculate the pathogen content based on the regression equation that was obtained.The concentrations of the DNA samples from the three genotypes  and XS2 were determined. The XS2 DNA solutions were subjected to six serial dilutions using sterilised water to obtain concentrations of 50, 5, 5 × 10 XS2 DNA . Using tThe leaves were collected from the plants after inoculation with the CBB pathogen, and the sampling periods were 0, 24, and 48 h post inoculation (hpi). Each genotype was examined three times, and a total of 27 samples were taken for transcriptome analysis. The samples were named with K for tepary bean PI 319443, R for HR45, and S for Bilu. The samples at 0 h were named K0-1, K0-2, K0-3, R0-1, R0-2, R0-3, S0-1, S0-2, and S0-3; the samples at 24 h were named K1-1, K1-2, K1-3, R1-1, R1-2, R1-3, S1-1, S1-2, and S1-3; and the samples at 48 h were named K2-1, K2-2, K2-3, R2-1, R2-2, R2-3, S2-1, S2-2, and S2-3. The total RNA from each sample was extracted using the Plant Total RNA Extraction Kit following the manufacturer’s instructions . The RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system  was used for accurate determination of the quantity and quality of the total RNA. The cDNA libraries of 27 high-quality RNA samples were constructed using the Illumina RNA-seq Kit and sequenced by the Illumina NovaSeq 6000 platform according to standard protocols, and 150-bp paired-end reads were generated. Clean reads were obtained by removing reads containing adaptors, reads containing poly-N sequences and low-quality reads from the raw data. Then, the Q20, Q30, and GC levels of the clean data were calculated. All downstream analyses were based on clean data with high quality.http://plants.ensembl.org/Phaseolus_vulgaris/Info/Index. The index of the reference genome was built using Hisat2 v2.0.5 . The p values were adjusted using the Benjamini and Hochberg method. A corrected p value of 0.05 and absolute fold change of 2 were set as the thresholds for significantly differential expression.Differential expression analysis was performed using the DESeq2 R package (1.20.0) . DESeq2 p values less than 0.05 were considered to be significantly enriched by DEGs. KEGG is a database resource for understanding the high-level functions and utilities of biological systems, such as cells, organisms and ecosystems, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies1. We used the cluster Profiler R package to test the statistical enrichment of DEGs in KEGG pathways (Gene Ontology (GO) enrichment analysis of DEGs was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected pathways .Weighted gene co-expression network analysis (WGCNA) is a systematic biological method used to describe the gene association modes among different samples. It can be used to identify gene sets that are highly synergistically changed and identify candidate biomarkers or therapeutic targets based on the coherence of gene sets and the correlation between gene sets and phenotypes. The R package WGCNA (version 3.6.1) is a set of functions used to calculate various weighted association analyses, which can be used for network construction, gene screening, gene cluster identification, topological feature calculation, data simulation, and visualisation .2O. The qRT-PCR experiment was repeated with three biological replicates for each sample, and the results were analysed by the 2Ct–ΔΔ method , respectively, while there was no significant difference between the amount of colonisation between PI 319443 and HR45 , which indicates that the amplification efficiency was close to the ideal state. Colonisation analysis showed that the amount of pathogenic colonisation in Bilu was 6.4 and 15.8 times higher than that in PI 319443 and HR45 . After filtering adaptors and low-quality reads, a total of 1,146,009,876 high-quality clean reads were generated, and each sample yielded approximately 6 Gb of clean reads . The ave10(FPKM+1) value, and the correlation coefficients  of the biological replicates were all above 95%, indicating a high degree of consistency among the replicates  structural domains, which are common in plant R genes, and receptor-like protein kinases, transcription factors, and oxidoreductases have also been reported to be associated with pathogen resistance, so these types of genes were the focus of this study. Eighteen resistance gene (R genes) containing LRR structural domains and nine genes containing protein kinase structural domains were identified. Nineteen DEGs belonging to the transcription factor family were also identified, encoding MYB, CPC, and bHLH transcription factors; 63 DEGs encoded oxidative factors, 37 were cytochrome P450-like DEGs, and 13 DEGs encoded peroxidase genes. Cytochrome P450 and peroxidase have been reported to have important roles in plant disease resistance.The 139 genes annotated from the 334 DEGs were categorised into three GO categories and 74 terms. Many genes were significantly enriched in biological processes associated with defence response, response to stress, response to biotic stimulus, response to oxidative stress, response to external stimulus, signal transduction, signalling, cell communication, cellular response to stimulus, regulation of response to stimulus, and multiorganism process. Furthermore, some GO terms were significantly enriched in molecular functions among the DEGs, including haem binding, iron ion binding, oxidoreductase activity, peroxidase activity, antioxidant activity, and ADP binding . KEGG anPhvul.001G160500, Phvul.006G106100, Phvul.007G127800), two jasmonate ZIM (JAZ) proteins , four secretory proteins (SCRs) , two Auxin , one kinase (Phvul.006G216400), and one bZIP_1 transcription factor (Phvul.009G026900). The transcription factors AP2 and bZIP_1 are important participants in the salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) signalling pathways, and they exercise an important function in the phytohormone-mediated signalling pathway for disease and stress resistance as key regulators of gene expression -type R genes containing the TIR (Toll/interleukin-1 receptor like) structural domain have an important role. TNL-like R genes are thought to be associated with the initiation of plant defence response signals and with pathogen recognition, and in structural studies of the flax stripe rust resistance gene L6, it was found that mutating amino acids in the conserved structural domain of TIR resulted in the disappearance of plant hypersensitive responses . There wl domain ; the comium wilt .2+ signalling pathway, hormone biosynthesis, oxidative burst and transcriptional reorganisation through crossover or specific signalling pathways (Phvul.006G216400) and one CDPK (Phvul.009G082532) were identified as upregulated DEGs, and one LRR-RLK (Phvul.007G265100) was identified as a downregulated DEG, in the three genotypes. KEGG analysis showed that three kinase genes were involved in “MAPK signalling pathway-plant” and “plant–pathogen interaction” pathways, indicating their important functions in Xap resistance.Protein kinases play crucial roles in plant resistance to pathogens. Plants recognise pathogens mainly through pattern-recognition receptors (PRRs) on the cell membrane surface, through microbe-associated molecular patterns (MAMPs), or through nucleotide-binding LRRs (NB-LRRs) as effector proteins. This process is involved in protein kinase activation, the Capathways . Among tPhvul.003G021000, Phvul.003G129200) and one JAR gene (Phvul.005G106500), acting as repressors of JA responses, were identified. Previous studies have shown that plants use JAR1 to synthesise JA-isoleucine (JA-Ile), the active form of the hormone, and subsequently recruit JAZ proteins to regulate the response of the JA gene and, in turn, the JA response. Furthermore, three AP2 transcription factors and one bZIP_1 transcription factor were found , cytokinin (CTK), and gibberellin (GA) to effectively activate the plant immune response. In this study, 17 DEGs were significantly enriched in the plant hormone signal transduction pathway. JA is a key endogenous growth regulator of plant defence responses to pathogenic microorganisms . Two JAZre found . The thrO-methyltransferases (Phvul.002G305200 and Phvul.003G242451) were homologous to ZmCCoAOMT2, which encodes a caffeoyl-CoAO-methyltransferase associated with the phenylpropanoid pathway and lignin production and confers resistance to both southern leaf blight and grey leaf spot  BioProject database under accession number JW and PY planned and designed the experiments. LW contributed to the lines breeding. PY performed the experiments of resistance evaluation. PY and YC analysed the data and wrote the manuscript. JW and SW revised the manuscript. All authors contributed to the article and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.