Systems Metabolic Engineering Meets Machine Learning: A New Era for Data‐Driven Metabolic Engineering

The recent increase in high‐throughput capacity of ‘omics datasets combined with advances and interest in machine learning (ML) have created great opportunities for systems metabolic engineering. In this regard, data‐driven modeling methods have become increasingly valuable to metabolic strain design. In this review, the nature of ‘omics is discussed and a broad introduction to the ML algorithms combining these datasets into predictive models of metabolism and metabolic rewiring is provided. Next, this review highlights recent work in the literature that utilizes such data‐driven methods to inform various metabolic engineering efforts for different classes of application including product maximization, understanding and profiling phenotypes, de novo metabolic pathway design, and creation of robust system‐scale models for biotechnology. Overall, this review aims to highlight the potential and promise of using ML algorithms with metabolic engineering and systems biology related datasets.     

Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells

The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral KCl cotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical and ovarian cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose- and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascades leading to the activation of Akt and extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways are differentially required for IGF-1-stimulated biosyn-thesis of KCC polypeptides. Specific reduction of Erk1/2 protein levels with small interference RNA abolishes IGF-1-stimulated KCC activity. Pharmacological inhibition and genetic modification of KCC activity demonstrate that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n = 28) and ovarian cancer (n = 35), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers.     

Identification of a basolateral Cl-/HCO3- exchanger specific to gastric parietal cells

The basolateral Cl(-)/HCO(3)(-) exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H(+)-K(+)-ATPase. Here, we report the identification of a new Cl(-)/HCO(3)(-) exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl(-)/HCO(3)(-) exchanger that is active in both acidic and alkaline pH(i). On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl(-)/HCO(3)(-) exchanger in gastric parietal cells and plays a major role in gastric acid secretion.     

Reconstruction of orbital floor fracture using solvent-preserved bone graft

The orbital floor is one of the most frequently damaged parts of the maxillofacial skeleton during facial trauma. Unfavorable aesthetic and functional outcomes are frequent when it is treated inadequately. The treatment consists of spanning the floor defect with a material that can provide structural support and restore the orbital volume. This material should also be biocompatible with the surrounding tissues and easily reshaped to fit the orbital floor. Although various autografts or synthetic materials have been used, there is still no consensus on the ideal reconstruction method of orbital floor defects. This study evaluated the applicability of solvent-preserved cadaveric cranial bone graft and its preliminary results in the reconstruction of the orbital floor fractures. Twenty-five orbital floor fractures of 21 patients who underwent surgical repair with cadaveric bone graft during a 2-year period were included in this study. Pure blowout fractures were determined in nine patients, whereas 12 patients had other accompanying maxillofacial fractures. Of the 21 patients, 14 had clinically evident diplopia (66.7 percent), 12 of them had enophthalmos (57.1 percent), and two of them had gaze restriction preoperatively. Reconstruction of the floor of the orbit was performed following either the subciliary or the transconjunctival approach. A cranial allograft was placed over the defect after sufficient exposure. The mean follow-up period was 9 months. Postoperative diplopia, enophthalmos, eye motility, cosmetic appearance, and complications were documented. None of the patients had any evidence of diplopia, limited eye movement, inflammatory reactions in soft tissues, infection, or graft extrusion in the postoperative period. Providing sufficient orbital volume, no graft resorption was detected in computed tomography scan controls. None of the implants required removal for any reason. Enophthalmos was seen in one patient, and temporary scleral show lasting up to 3 to 6 weeks was detected in another three patients. Satisfactory cosmetic results were obtained in all patients. This study showed that solvent-preserved bone, which is a nonsynthetic, human-originated, processed bioimplant, can be safely used in orbital floor repair and can be considered as another reliable treatment alternative.     

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

Although K-Cl cotransporter (KCC1) mRNA is expressed in manytissues, K-Cl cotransport activity has been measured in few cell types,and detection of endogenous KCC1 polypeptide has not yet been reported.We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flankinggenomic regions and mapped the mKCC1 gene to chromosome 8. Threeanti-peptide antibodies raised against recombinant mKCC1 function asimmunoblot and immunoprecipitation reagents. The tissue distributionsof mKCC1 mRNA and protein are widespread, and mKCC1 RNA isconstitutively expressed during erythroid differentiation of ES cells.KCC1 polypeptide or related antigen is present in erythrocytes ofmultiple species in which K-Cl cotransport activity has beendocumented. Erythroid KCC1 polypeptide abundance is elevated inproportion to reticulocyte counts in density-fractionated cells, inbleeding-induced reticulocytosis, in mouse models of sickle celldisease and thalassemia, and in the corresponding human disorders.mKCC1-mediated uptake of ⁸⁶Rb intoXenopus oocytes requires extracellularCl, is blocked by thediureticR(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling,N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

     

Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl^- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca²⁺-dependent Cl^- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca²⁺ transients via activation of a long-lived, store-operated Ca²⁺ entry pathway. The enhanced intracellular Ca²⁺ signal is associated with uncoupling of the Cl^- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca²⁺ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects. nelfinavir; clotrimazole; barium     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Association of polymorphisms in the HLA-B region with extended haplotypes

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLa-B, respectively. Five different RFLP variants occuring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30, DR3], [HLa-Bw62,SC33,Dr4], [HLa-B44,SC30,Dr4], and [HLA-Bw47,FC91,0DR7]. The patterns of 6.9 kb/ 3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21, DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was suprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.