Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

Breastfeeding during a Pandemic.

BACKGROUND: The current pandemic and the concerns of vertical transmission of SARS-CoV-2 have contributed to increasing the rate of breastfeeding interruption. This tendency has been associated with negative effects on the well-being of lactating mothers and their infants. The aim of this review is to summarize the evidence on the strategies to support breastfeeding during the COVID-19 pandemic and on the safety of breastfeeding during a SARS-CoV-2 infection or after COVID-19 vaccination. SUMMARY: Available data show that the lack of support of lactating mothers during the pandemic has contributed to breastfeeding cessation worldwide. However, a few strategies have been proposed to overcome this issue. The risk of SARS-CoV-2 transmission from infected mothers to their offspring is extremely low. Furthermore, vaccination of lactating mothers is not associated with side effects in their infants. Key Messages: Increasing effort should be made to support breastfeeding during the COVID-19 pandemic. Mothers who are able to take care of their offspring and to adopt basic hygiene measures should not interrupt breastfeeding during a SARS-CoV-2 infection. Vaccination of lactating mothers might further strengthen the protective effect of breastfeeding against infections.

A light-gated potassium channel for sustained neuronal inhibition.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins.

Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.

The masks of Lorenzo Tenchini: their anatomy and surgical/bioengineering clues.

An academic, anatomist, and Lombrosian psychiatrist active at the University of Parma in Italy at the end of the 19th century, Lorenzo Tenchini produced ceroplastic-like masks that are unique in the anatomical Western context. These were prepared from 1885 to 1893 with the aim of 'cataloguing' the behaviour of prison inmates and psychiatric patients based on their facial surface anatomy. Due to the lack of any reference to the procedure used to prepare the masks, studies were undertaken by our group using X-ray scans, infrared spectroscopy, bioptic sampling, and microscopy analysis of the mask constituents. Results showed that the masks were stratified structures including plaster, cotton gauze/human epidermis, and wax, leading to a fabrication procedure reminiscent of 'additive layer manufacturing'. Differences in the depths of these layers were observed in relation to the facial contours, suggesting an attempt to reproduce, at least partially, the three-dimensional features of the facial soft tissues. We conclude the Tenchini masks are the first historical antecedent of the experimental method for face reconstruction used in the early 2000s to test the feasibility of transferring a complete strip of face and scalp from a deceased donor to a living recipient, in preparation for a complete face transplant. In addition, the layering procedure adopted conceptually mimics that developed only in the late 20th century for computer-aided rapid prototyping, and recently applied to bioengineering with biomaterials for a number of human structures including parts of the skull and face. Finally, the masks are a relevant example of mixed ceroplastic-cutaneous preparations in the history of anatomical research for clinical purposes.

HCN1 mutation spectrum: from neonatal epileptic encephalopathy to benign generalized epilepsy and beyond.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control neuronal excitability and their dysfunction has been linked to epileptogenesis but few individuals with neurological disorders related to variants altering HCN channels have been reported so far. In 2014, we described five individuals with epileptic encephalopathy due to de novo HCN1 variants. To delineate HCN1-related disorders and investigate genotype-phenotype correlations further, we assembled a cohort of 33 unpublished patients with novel pathogenic or likely pathogenic variants: 19 probands carrying 14 different de novo mutations and four families with dominantly inherited variants segregating with epilepsy in 14 individuals, but not penetrant in six additional individuals. Sporadic patients had epilepsy with median onset at age 7 months and in 36% the first seizure occurred during a febrile illness. Overall, considering familial and sporadic patients, the predominant phenotypes were mild, including genetic generalized epilepsies and genetic epilepsy with febrile seizures plus (GEFS+) spectrum. About 20% manifested neonatal/infantile onset otherwise unclassified epileptic encephalopathy. The study also included eight patients with variants of unknown significance: one adopted patient had two HCN1 variants, four probands had intellectual disability without seizures, and three individuals had missense variants inherited from an asymptomatic parent. Of the 18 novel pathogenic missense variants identified, 12 were associated with severe phenotypes and clustered within or close to transmembrane domains, while variants segregating with milder phenotypes were located outside transmembrane domains, in the intracellular N- and C-terminal parts of the channel. Five recurrent variants were associated with similar phenotypes. Using whole-cell patch-clamp, we showed that the impact of 12 selected variants ranged from complete loss-of-function to significant shifts in activation kinetics and/or voltage dependence. Functional analysis of three different substitutions altering Gly391 revealed that these variants had different consequences on channel biophysical properties. The Gly391Asp variant, associated with the most severe, neonatal phenotype, also had the most severe impact on channel function. Molecular dynamics simulation on channel structure showed that homotetramers were not conducting ions because the permeation path was blocked by cation(s) strongly complexed to the Asp residue, whereas heterotetramers showed an instantaneous current component possibly linked to deformation of the channel pore. In conclusion, our results considerably expand the clinical spectrum related to HCN1 variants to include common generalized epilepsy phenotypes and further illustrate how HCN1 has a pivotal function in brain development and control of neuronal excitability.

The first Industrial Hygiene Congresses in Milan.

The first Congress of the Italian Association of Industrial Hygienists was held in 1977. Nevertheless, at the end of the 19th century some issues concerning industrial hygiene had been discussed during the 3rd International Congress on Accidents and Social Insurance (Milan, October 1-6, 1894). A year later, the Milanese trade Unions held another Congress on this theme. In 1902 Leopoldo Sconfietti, an engineer, proposed a new system of air conditioning to be used in textile factories. After the EXPO 1906 in Milan, a museum devoted to Industrial Hygiene was opened in the city, but unfortunately it is now lost. Soon after the opening in 1910 of the Milanese "Clinica del Lavoro", the oldest Occupational Health Institute in the world, an International technical Congress devoted to Industrial Hygiene was held in Milan in May 1912, when the discipline became autonomous from Occupational Medicine and Social Insurance.

Benzene and leukemia: from scientific evidence to regulations. A historical example.

BACKGROUND: Benzene is a highly flammable, highly volatile liquid aromatic hydrocarbon. It has been used in many industrial processes as a solvent or a starting material. At the beginning of the twentieth century, it was very widely used in the workplace, especially in printing and in the shoe manufacturing and rubber industries. Although benzene was first recognized to cause aplastic anemia, its association with leukemia has been investigated only since the 1930s. In 1963, Italy was one of the first countries in the world to adopt a law to ban benzene as a solvent in work activities. OBJECTIVES: This study analyzed the contribution of the Clinica del Lavoro in Milan, Italy, to studies of the relationship between exposure to benzene and leukemia. METHODS: Scientific literature and historical sources on benzene and leukemia in the twentieth century were reviewed, and interviews with a first-hand witness of that period were conducted. RESULTS: By 1928, several scholars had reported anecdotal cases of leukemia among workers exposed to benzene. Enrico Vigliani was the first to collect all of these cases and to try to conduct statistical analysis on these data, in order to support the association between benzene and leukemia. In the 1960s, Vigliani and Alessandra Forni showed that benzene could cause chromosome aberrations in the bone marrow that could produce leukemic clones. CONCLUSIONS: As a result of these studies and the subsequent regulations which banned benzene, exposure conditions changed in the workplace in the last few decades. The resulting low concentrations have prompted researchers to investigate new exposure biomarkers and to study any related health problems.

Occupational medicine and occupational psychology: a rendezvous in Milan after World War II.

BACKGROUND: In the 1950s, occupational medicine and occupational psychology found a common field of action in the Clinica del Lavoro in Milan. OBJECTIVES This study aims to analyze and document how this encounter took place and, in particular, the contribution of the Clinica del Lavoro to the development of occupational psychology in Italy. METHODS: Historical sources of that period were investigated. RESULTS: Before the 1950s, experimental psychology was only taught at the Catholic University of the Sacred Heart in Milan. The first professor of clinical psychology in the School of Medicine at the State University of Milan, was Marcello Cesa-Bianchi (1926-2018). He collaborated with the graphic, textile and pharmaceutical industries for the personnel training and management, and carried out important research in occupational psychology on behalf of the European Coal and Steel Community. The Chair of Clinical Psychology was initially located in the Clinica del Lavoro and the activity of the team of Cesa-Bianchi was oriented towards the elaboration of professional profiles and job analysis. In those years Cesa-Bianchi also conducted pioneering research in the field of psycho-gerontology. CONCLUSIONS: The historical experience that integrated psychology and occupational medicine in the scientific context of Milan contains a series of values, useful to today's reflection and practice. Our work also undelines the importance of preserving historical documents: only a better knowledge of history can guarantee a better destiny.

[Mobile miniature X-ray evaluation and pneumoconiosis: the role of the Clinica del Lavoro in Milan (1941-1948)]. / La valutazione schermografica delle pneumoconiosis (1941-1948): il ruolo della Clinica del Lavoro di Milano.

Since the end of the 19th century, X-rays have been used to detect lung diseases. In Italy, 207,096 miniature chest radiographs were taken from 1941 to 1948. Traditional radiographs gave better results, but miniature chest radiographs were useful for screening. Indeed, the development of mobile miniature chest radiography units resulted in an improvement in mass X-rays screening for the detection of penumoconiosis. These mobile miniature units were mounted on a bus chassis, a solution that allowed to easily reach workers. The authors analyze some models of X-ray wagon units used by the "Clinica del Lavoro" in Milan in the 1950s. From the point of view of medical museology, the preservation of these devices requires appropriate spaces.

Work and health care in Milan, 1864-1874. / Lavoro e assistenza sanitaria a Milano, 1864-1874.

In 1864 in Milan, Giovanni Rosmini (1832-1896) opened an ophthalmic dispensary for workers. In 1874 this dispensary was transformed into the first ophthalmic hospital of the city. This hospital still exists today. The authors analyze a document that belonged to the lawyer Enrico Rosmini (1828-1898), brother of Giovanni, which helps to piece together the early years of the dispensary, where about 4,000 surgeries were carried out in the first four years of activity. This historical document is valuable as it sheds light on one of the first healthcare institution for workers in Milan.

Between Bouillaud and Broca: An unknown Italian debate on cerebral localization of language.

From 1825 onward, Bouillaud began gathering clinical evidence to support the hypothesis that speech is located in the cerebral frontal lobes. His aim was to provide empirical proof to Gall's theory of a specific substratum of speech in the anterior region of the brain. A well-known discussion ensued inside the French school among supporters and detractors that went far beyond Broca's first report in 1861. Unknown is that Bouillaud's investigations on localization of articulated language also gave rise to a discussion in Italy in the same period. In particular, speech localization formed a central topic in the mid-19th century in Northern Italy mainly thanks to four physicians, Michelangelo Asson, Mosè Rizzi, Gaetano Strambio and Filippo Lussana, who reported on language-impaired patients and approached these cases in the light of Bouillaud's claims. Similarly to the French debate, the Italian medical community also included attacks and advocacies of the hypothesis of a precise localization of articulated language in the frontal lobes. However, they were mainly interested in investigating the anatomo-clinical relationships rather than in supporting Gall's organology. This Italian debate appears to be the first to have developed in the mid-19th century outside that of the French community.

[Industrial first aid equipment: a historical analysis (1840-1914) ]. / Gli strumenti di pronto soccorso in azienda: un'analisi storica (1840-1914).

Even if references to the tools required to intervene after an accident can be found in the works of Bernardino Ramazzini (1633-1714) or Johann Peter Frank (1745-1821), it was only with the development of industrial manufacturing that the need to study means to prevent and intervene in cases of accident became evident. In October 1894 the III Congrés International des Accidents du Travail et des Assurances Sociales was held in Milan. The following year, the Milanese trade union movement acknowledged the necessity to address the problem of industrial accidents. In 1896 the Association for Medical Assistance in  Industrial Accidents was founded in Milan. A specific medical institute was set up, appropriate first aid tools were collected and first aid rooms in the main Milanese factories were inaugurated. Nevertheless, few data seem to be available regarding the manufacture and use of this equipment in industry. We analyzed more than fifty catalogs of European industrial products, between 1843 and 1914, to study the evolution of first aid equipment for industrial use. They reflect and attest to the evolution of medicine and surgery, although some models seem to be related to certain industrial categories (railways, electrical appliances), some were similar to ordinary first aid boxes, others were strictly related to surgery; some could only be used by physicians, and others only by workers. Identification, conservation, and reappraisal of these tools is essential for historians of occupational health because these objects were normally not preserved. The catalogues of industrial production are also precious sources, since they are rarely preserved in public libraries and deserve to be used for historical studies.

A high affinity switch for cAMP in the HCN pacemaker channels.

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Large-scale production of polyimide micropore-based flow cells for detecting nano-sized particles in fluids.

In diagnostic and sequencing applications, solid-state nanopores hold significant promise as a single-molecule sensing platform. The fabrication of precisely sized pores has traditionally been challenging, laborious, expensive, and inefficient, which has limited its applications until recently. To overcome this problem, this paper proposes a novel, reliable, cost-effective, portable, mass-productive, robust, and ease-of-use micropore flow cell that works based on the resistive pulse sensor (RPS) technique in order to distinguish the different sizes of c nanoparticles. RPS is a robust and informative technique that can provide valuable details of the size, shape, charge, and individual particle concentrations in the media. By femtosecond laser drilling of a polyimide substrate as an alternate material, translocation of 100, 300, and 350 nm polystyrene nanoparticles in PBS buffer was distinguished by 0.1, 1, and 2 nA current blockade levels, respectively. This is the first time a micropore has been opened in a polyimide membrane using a femtosecond laser in a single step. The experimental and theoretical investigation, scanning electron microscopy and focused ion beam spectroscopy were performed to comprehensively explain the micropore's performance. We showed that our innovative micropore-based flow cell could distinguish nano-sized particles in fluids, and it can be used in large-scale production because of its simplicity and cost-effectiveness.

Corrigendum: Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements.

[This corrects the article DOI: 10.3389/fphys.2022.998176.].

Papaver somniferum in seventeenth century (Italy): archaeotoxicological study on brain and bone samples in patients from a hospital in Milan.

In this paper, we present the results of toxicological analyses of preserved brain tissue and bone samples from the remains of the seventeenth century patients of the Ospedale Maggiore, the main hospital in Milan and one of the most innovative hospitals in Europe from the Renaissance period. Beneath it, the crypt functioned as the burial place for the deceased of the hospital. In this multidisciplinary study of the remains, toxicological analyses in particular were performed with HPLC-MS/MS on different biological samples from nine individuals. Anthropological, paleopathological, histological, radiological examinations and radiocarbon dating were also carried out. As a result, archeotoxicological analyses revealed the presence of codeine, morphine, noscapine and papaverine, derived from Papaver somniferum, a plant present in the hospital pharmacopeia used as a narcotic, analgesic, astringent, coagulant, and antitussive agent. Such analyses have shed light on the pharmacological therapies administered to the patients near the time of death and have implemented our knowledge of medical treatment and drug administration in the 1600's.

Filippo Lussana (1820-1897): from medical practitioner to neuroscience.

Filippo Lussana's scientific activities are neglected in neurological field. His activity could be divided into two phases: as a medical practitioner and the academic period, as a clinical neurophysiologist. The focus of his various research studies was the central nervous system, including balance disorders (the role of the cerebellum and semicircular canals), taste innervation, pain and speech disorders (theories of brain localisation). Lussana's clinical method and direct dealings with patients laid the groundwork for his subsequent academic activities as a neurophysiologist, in Parma and later in Padua. Influenced by phrenological theories, he also conducted interesting studies on synaesthesia for which he determined a brain site. He contributed to the neurophysiology of taste, emphasising the role of the facial nerve. With his study on muscle sense, he also joined the debate on the role of the cerebellum in balance, setting the foundation for later studies on this organ by Luigi Luciani.

The Big Game: hunting for retained bullets and foreign bodies in Great War radiology.

OBJECTIVES: Searching for retained bullets has always been crucial in war surgery. Aim of this paper is to briefly outline the history of retained bullet identification methods before X-rays discovery and describe the proliferation of the most significant methods of foreign body localization during WWI. METHODS: Coeval medical journals, reference textbooks, dedicated manuals and documents have been searched and compared in multiple archives and on the internet. RESULTS: Before radiologic era, probing the wound was the only way to detect the bullet and minimize the need of a large surgical incision (anaesthesia was walking its first tentative steps and antisepsis still to be conceived). Nelaton's probe, specifically designed to detect General Garibaldi's retained projectile, gained popularity. Application of electricity provided further rudimental aids to find retained metals. X-rays discovery made bullet detection easy, but exact localization to guide removal was still difficult. Hundreds of imaginative X-Ray methods for localizing bullets and splinters more precisely in the countless complex wounds flourished during the Great War. Axis intersection, geometric reconstruction and anatomical criteria guided localization. Complex procedures and rudimental localizers to simplify calculations, and a number of compasses and magnetic or electric devices to aid surgical removal were developed, and are here outlined. Intermittent radiology assessment or combined radiology and surgery procedures started to play a role. CONCLUSIONS: All these methods and tools are the ancestors of modern navigation systems, ensured by images digitalization and miniaturization technologies. KEY WORDS: Foreign bodies, Mobile Health Units, Radiology, X-Rays, World War I, Wounds and injuries.