The flexibility of ACE2 in the context of SARS-CoV-2 infection.

The COVID-19 pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine developments efforts are underway, many questions remain outstanding on the mechanism of SARS-CoV-2 viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and entry in the cell. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular Spike glycoprotein and at the receptor binding domain-receptor interface, suggesting a role in infection. Here, we perform all-atom molecular dynamics simulations of the glycosylated, full-length membrane-bound ACE2 receptor, in both an apo and spike receptor binding domain (RBD) bound state, in order to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix, while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer, and suggests a mechanical contribution of the host receptor towards the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can be exploited for the rational design of effective SARS-CoV-2 therapeutics. STATEMENT OF SIGNIFICANCE: As the host receptor of SARS-CoV-2, ACE2 has been the subject of extensive structural and antibody design efforts in aims to curtail COVID-19 spread. Here, we perform molecular dynamics simulations of the homodimer ACE2 full-length structure to study the dynamics of this protein in the context of the cellular membrane. The simulations evidence exceptional plasticity in the protein structure due to flexible hinge motions in the head-transmembrane domain linker region and helix mobility in the membrane, resulting in a varied ensemble of conformations distinct from the experimental structures. Our findings suggest a dynamical contribution of ACE2 to the spike glycoprotein shedding required for infection, and contribute to the question of stoichiometry of the Spike-ACE2 complex.

A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.

Heterodimerization with Fra-1 cooperates with the ERK pathway to stabilize c-Jun in response to the RAS oncoprotein.

Multiple tumorigenic pathways converge on the activating protein-1 (AP-1) family of dimeric transcription complexes by affecting transcription, mRNA decay, posttranslational modifications, as well as stability of its JUN and FOS components. Several mechanisms have been implicated in the phosphorylation- and ubiquitylation-dependent control of c-Jun protein stability. Although its dimer composition has a major role in the regulation of AP-1, little is known about the influence of heterodimerization partners on the half-life of c-Jun. The FOS family member Fra-1 is overexpressed in various tumors and cancer cell lines wherein it controls motility, invasiveness, cell survival and cell division. Oncogene-induced accumulation of Fra-1 results from both increased transcription and phosphorylation-dependent stabilization of the protein. In this report, we describe a novel role of Fra-1 as a posttranslational regulator of c-Jun. By using both constitutively and inducible transformed rat thyroid cell lines, we found that c-Jun is stabilized in response to RAS oncoprotein expression. This stabilization requires the activity of the extracellular signal-related kinase (ERK) pathway, along with c-Jun heterodimerization with Fra-1. In particular, heterodimerization with Fra-1 inhibits c-Jun breakdown by a mechanism dependent on the phosphorylation of the Fra-1 C-terminal domain that positively controls the stability of the protein in response to ERK signaling. Therefore, Fra-1 modulates AP-1 dimer composition by promoting the accumulation of c-Jun in response to oncogenic RAS signaling.

[Selection of candidates for cardiac resynchronisation therapy and prediction of their response.]. / Selezione dei candidati alla terapia di resincronizzazione cardiaca e predizione della loro risposta.

AIMS: Cardiac resynchronization therapy is currently used in selected patients with end-stage heart failure. However, 30% of patients do not respond to CRT. The aim of our study was to find echocardiographic (TDI), electrocardiographic (QRS interval and electric distance between right and left catheter), clinical (6MW test) or autonomical (HRV) parameters able to predict responsiveness to CRT. MATERIALS AND METHODS: 47 patients (mean age 74+/-10 years) with end-stage heart failure, symptomatic, with left ventricular (LV) ejection fraction less than 35% and QRS 120 ms, underwent CRT. RESULTS: At thirteen months follow up, all clinical and echocardiographic parameters significantly improves (EF p<0.001; LVED volume p<0.001; 6MWT p<0.001; max delay TDI p<0.001; HRV p<0.05; Right-left distance p<0.05). A positive response was documented in 31/47 (67.4%) patients who presented an increase in LVEF > or = 5 units. There was a significant difference of LVED diameter (p<0.05) and HRV (p<0.05) between responders and non responders. Receiver-operating curve analysis showed that a positive response to CRT may be predicted in patients with LVED diameter <67 mm (with a sensitivity of 77% and a specificity of 88%). CONCLUSIONS: Our results confirm the clinical improvement obtained by CRT in end-stage heart failure patients as well as the limited value of QRS duration and intraventricular dyssynchrony as predictor of clinical recovery after CRT. While a most-advanced clinical stage of disease (HRV) without an advance left ventricular remodeling (LVED diameter) demonstrated to predict response to CRT, with sensitivity of 77% and specificity of 88%.

An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation.

The transcription factor AP-1 plays key roles in tumorigenesis, by regulating a variety of protein-coding genes, implicated in multiple hallmarks of cancer. Among non-coding genes, no AP-1 target has been described yet in tumorigenesis. MicroRNAs (miRNAs) are negative post-transcriptional regulators of protein-coding genes. miRNA expression signatures are highly relevant in cancer and several tumor-associated miRNAs (oncomirs) play critical roles in oncogenesis. Here, we show that the miRNA miR-21, which represents the most frequently upregulated oncomir in solid tumors, is induced by AP-1 in response to RAS. By analyzing validated miR-21 targets, we have found that the tumor suppressors PTEN and PDCD4 are downregulated by RAS in an AP-1- and miR-21-dependent fashion. We further show that, given the role of PDCD4 as negative regulator of AP-1, the miR-21-mediated downregulation of PDCD4 is essential for the maximal induction of AP-1 activity in response to RAS. Our data reveal a novel mechanism of positive autoregulation of the AP-1 complex in RAS transformation and disclose the function of oncomirs as critical targets and regulators of AP-1 in tumorigenesis.

Primary percutaneous transluminal coronary angioplasty in the setting of multivessel disease.

Primary percutaneous coronary intervention of the culprit lesion is the treatment of choice for acute myocardial infarction, while treatment of the severe non culprit lesion is not indicated in the guidelines (Class III indication). More aggressive strategies that include initial treatment of the severe non culprit lesion may reduce the incidence of delayed occlusions in specific clinical settings. The two cases we describe support our point of view.

Canaries in a coal mine: California physician groups and competition.

Health care organizations may compete by developing organized processes to improve quality and increase efficiency, or may focus on growing to increase negotiating leverage and on controlling costs through withholding appropriate care and avoiding sick patients. This paper describes key ways in which public and private policy decisions create incentives that influence the competitive focus of physician groups in California, a state in which physician groups and health maintenance organizations are prevalent. These policies do not manage competition in optimal ways: They reward groups for market leverage and controlling costs while failing to fully reward quality and efficiency.

Role of distinct mitogen-activated protein kinase pathways and cooperation between Ets-2, ATF-2, and Jun family members in human urokinase-type plasminogen activator gene induction by interleukin-1 and tetradecanoyl phorbol acetate.

We have investigated the in vivo and in vitro regulation of the human urokinase-type plasminogen activator (uPA) gene by interleukin-1 (IL-1) and analyzed the transcription factors and signalling pathways involved in the response of the -2.0-kb uPA enhancer to IL-1 induction and to tetradecanoyl phorbol acetate (TPA) induction. Mutational analysis showed the cooperative activity of the Ets-binding site (EBS) and the two AP-1 elements of the enhancer. The results reveal that the EBS is required for the response to both inducers mediated by Ets-2, which is regulated at a level subsequent to DNA binding, by an IL-1- and phorbol ester-inducible transactivation domain. Both the IL-1 and the TPA-mediated induction result in a drastic increase of AP-1 binding to the downstream site of the enhancer (uPA 3' TPA-responsive element), while a mostly qualitative change, resulting from the interplay between ATF-2 homodimers and c-Jun-ATF-2 heterodimers, takes place at the upstream AP-1 element. The analysis of two distinct mitogen-activated protein kinase pathways shows that stress-activated protein kinase-Jun N-terminal kinase activation, resulting in the phosphorylation of ATF-2, c-Jun, and JunD, is required not only for the IL-1- but also for the TPA-dependent induction, while the extracellular signal-related kinase 1 (ERK-1) and ERK-2 activation is involved in the TPA- but not in the IL-1-dependent stimulation of the uPA enhancer.

Primary care physicians should be coordinators, not gatekeepers.

Primary care gatekeeping, in which the goal of the primary care physician (PCP) is to reduce patient referrals to specialists and thereby reduce costs, is not an adequate system in which to practice medicine. However, returning to the pre-managed care model of uncoordinated open access to specialists is a poor solution. The primary care model should be retained, but PCPs should be transformed from gatekeepers into coordinators of care, in which the goal of the PCP is to integrate both primary and specialty care to improve quality. Changes in the PCP's daily work process, as well as the referral and payment processes, need to be implemented to reach this goal. This model would eliminate the requirement that referrals to specialists be authorized by the primary care physician or managed care organization. Financial incentives would be needed, eg, to encourage PCPs to provide management of complex cases and discourage both over-referral and underreferral to specialists. Budgeting specialists should control excess costs that might be created by the elimination of the primary care gatekeeper. Pilot projects are needed to test and refine this model of PCP as coordinator of care.

Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression.

We have investigated the role of the NFkappaB complex in the process of thyroid carcinogenesis by analysing thyroid carcinoma cell lines. A significant increase in p65 NFkappaB mRNA and protein expression, compared to normal thyroid cultures or tissue, was found in all of the cancer cell lines. Conversely, only a modest increase in the p50 NFkappaB mRNA and protein was found in most, but not all carcinoma cell lines. The block of p65 protein synthesis with specific antisense oligonucleotides greatly reduced the ability of two undifferentiated carcinoma cell lines to form colonies in agar and reduced their growth rate. On the other hand, no effect was observed in the same cell lines when treated with p50 specific antisense oligonucleotides. These inhibitory effects seem to be mediated by the suppression of c-myc gene expression, since treatment with antisense oligonucleotides for p65 gene interfered negatively with c-myc gene expression. Our results indicate that activation of the NFkappaB complex by overexpression of p65 plays a critical role in the process of thyroid cell transformation.

Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

The expression of the high mobility group I (HMGI)-C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI-C gene product, we have analyzed the AP-1 complex in normal, fully transformed and antisense HMGI-C-expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP-1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra-1 gene induction, which is prevented in cell lines expressing the antisense HMGI-C. These results indicate that the HMGI-C gene product is essential for the junB and fra-1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra-1 protein synthesis by stable transfection with a fra-1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra-1 gene product in the process of cellular transformation.

NF-kappa B-mediated regulation of urokinase gene expression by PMA and TNF-alpha in human A549 cells.

Binding sites for different transcription factors have been identified in the regulatory region of the human uPA gene. We investigated the role of NF-kappa B and AP-1 families of transcription factors in the induction of uPA mRNA by TNF-alpha and PMA in the A549 cell line constitutively expressing uPA mRNA and protein. Using the protein synthesis inhibitor cycloheximide and the antioxidant PDTC, that have an opposite effect on NF-kappa B and AP-1 activation, we showed that uPA mRNA induction by TNF-alpha and PMA in the A549 cell line is mainly due to NF-kappa B activation.

Vertical integration and organizational networks in health care.

This paper documents the growing linkages between primary care-centered medical groups and specialists and between physicians and hospitals under managed care. We evaluate the two alternative forms of organizational coordination: "vertical integration," based on unified ownership, and "virtual integration," based on contractual networks. Excess capacity and the need for investment capital are major short-term determinants of these vertical versus virtual integration decisions in health care. In the longer term, the principal determinants are economies of scale, risk-bearing ability, transaction costs, and the capacity for innovation in methods of managing care.

The growth of medical groups paid through capitation in California.

BACKGROUND: In California, it is common for health maintenance organizations (HMOs) to contract with large medical groups that are paid through capitation and are responsible for managing a full spectrum of medical services. METHODS: We studied six large medical groups in California--Bristol Park Medical, Friendly Hills HealthCare Network, HealthCare Partners Medical Group, Mullikin Medical Centers, Palo Alto Medical Foundation, and San Jose Medical Group--that are paid through capitation and that are growing as a result of contracts with managed-care organizations. We conducted interviews and obtained data on factors such as patient enrollment, capitation and other revenue, numbers of days spent by enrollees in the hospital, and numbers of visits to physicians per enrollee. RESULTS: Between 1990 and 1994, the number of HMO enrollees whose care was paid for through capitation in the six medical groups increased by 91 percent, from 398,359 to 759,474. In 1994, the mean number of hospital days per 1000 HMO enrollees ranged from 120 to 149 for non-Medicare patients and from 643 to 936 days for Medicare patients. By comparison, in 1993 the mean numbers of hospital days per 1000 HMO enrollees not covered by Medicare were 232 for California and 297 for the United States; for HMO enrollees covered by Medicare, the numbers were 1337 for California and 1698 for the United States. In 1994, the average annual number of visits to physicians for HMO patients in the six groups not covered by Medicare ranged from 3.1 to 3.9; for Medicare patients, it ranged from 7.2 to 9.3; these rates were slightly lower than statewide and national rates. Four of the groups have sold their assets (such as facilities, supplies, equipment, and patients' charts) to outside investors; the physicians remain employed by physician-owned professional corporations. CONCLUSIONS: Medical groups paid through capitation offer a model for the status of physicians in managed-care systems that differs from the employee status offered by staff-model HMOs and the subcontractor status offered by HMOs that negotiate directly with individual physicians. Despite their growth, such medical groups in California face substantial challenges, such as obtaining the financial assets necessary to sustain rapid growth.

Balancing incentives. How should physicians be reimbursed?

The May 15, 1991, JAMA theme issue presented 14 different proposals for reforming the US medical care system. Though these proposals varied widely, they encompassed only three proposed methods for paying physicians: fee-for-service, salary, and capitation. When examined from a perspective that considers the incentives affecting practicing physicians, each of these methods has serious flaws. I outline these flaws and suggest an alternative method for reimbursing primary care physicians: that of combining capitation and fee-for-service. Specialists would be paid fee-for-service, but the cost of specialty care would be limited by giving each primary care physician a budget for such care. Consistent failure by a physician to stay within the budget would result in ongoing review, but not in direct financial sanctions. This system of reimbursing physicians is intended to balance incentives so that physicians can concentrate on making medical decisions with a minimum of distraction by third parties and by personal financial considerations.