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.

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.

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.

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

Tetratricopeptide repeat-containing Rab8b-interacting (TRIP8b) protein is a brain-specific subunit of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, a class of voltage-gated channels modulated by cyclic nucleotides. While the interaction between TRIP8b and the cytosolic C terminus of the channel has been structurally described, the HCN:TRIP8b stoichiometry is less characterized. We employed single molecule mass photometry (MP) to image HCN4 particles purified in complex with TRIP8b. Our data show that four TRIP8b subunits are bound to the tetrameric HCN4 particle, confirming a 1:1 stoichiometry.

Seizures, behavioral deficits, and adverse drug responses in two new genetic mouse models of HCN1 epileptic encephalopathy.

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the human HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool toward reaching this objective.

The Moro reflex: insights into the pathophysiology of generalized tonic-clonic seizures and infantile spasms

The Moro reflex (MR) is a primitive reflex that disappears after the first three months of life. It was described by the Austrian paediatrician Ernst Moro (1874-1951) in 1918, although the earliest visual representation of the MR dates back to the first half of the 14th Century, in a fresco by Ambrogio Lorenzetti (1290-1348). The neural centre underlying the MR is located in the lower part of the brainstem since it can be elicited also in anencephalic infants, as shown by the Austrian neurologist Eduard Gamper (1887-1938) in the first medical description of anencephaly (1926). The MR is due to the activation of an archaic neural circuit present in the newborn, the activity of which is later inhibited by the upper brain structures. Given their semiological resemblance, epileptic spasms and generalized tonic-clonic seizures might be due (at least partly) to the pathological activation of the same neural archaic circuit involved in the genesis of the MR. The neuronal network underlying these different phenomena might be located in the pons. In these seizure types, the activation of the same neural circuitry involved in the MR could occur through either direct excitation or through an indirect "liberating" mechanism, secondary to epileptic disruption of cortical inhibitory control on subcortical structures. The movements of the upper extremities in epileptic spasms, in the initial phase of generalized tonic-clonic seizures, and the MR might involve a distinct neural circuitry, which is (or becomes) hyperexcitable as a consequence of a pathological condition (epilepsy) or physiological brain immaturity (the MR).

The first major vaccination campaign against smallpox in Lombardy: the mass vaccination campaign against coronavirus nothing new only terminology.

Terminology, technology, communication and organizational strategies are different but we tried to compare the mass vaccination campaign against smallpox with the one we are doing against coronavirus.

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.

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.

Do the functional properties of HCN1 mutants correlate with the clinical features in epileptic patients?

The altered function of the Hyperpolarization-activated Cyclic-Nucleotide-gated (HCN) ion channels plays an important role in the pathogenesis of epilepsy in humans. In particular, HCN1 missense mutations have been recently identified in patients with different epileptic phenotypes, varying from mild to severe. Their electrophysiological characterization shows that mutated channels can act both with loss-of-function and gain-of-function mechanisms of action, without an evident correlation with the phenotype. In search for a correlation between clinical features and biophysical properties of the mutations, in this work we considered sixteen HCN1 mutations, found in eighteen Early Infantile Epileptic Encephalopathy (EIEE) patients. Statistical analysis did not establish any significant correlation between the clinical parameters and the current properties of the mutant channels. The lack of significance of our results could depend on the small number of mutations analyzed, epilepsy-associated with certainty. With the progressive increase of Next Generation Sequencing in patients with early-onset epilepsy, it is expected that the number of patients with HCN1 mutations will grow steadily. Functional characterization of epilepsy-associated HCN1 mutations remains a fundamental tool for a better understanding of the pathogenetic mechanisms leading to the disease in humans.

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.

Reports on Greenlandic and Faroese medicine from an eighteenth-century medical book.

The authors analyse descriptions of Greenlandic and Faroese medicine found in an Italian medical publication from the 18th century entitled, Europae Medicina a Sapientibus Illustrata […], which was printed in Brescia, in Northern Italy, in 1747. The author of these descriptions, Francesco Roncalli Parolino (1692-1769), was a renowned European physician. Roncalli Parolino focused his study on the treatment of scurvy and he promoted the inclusion of the Greenlandic and Faroese therapy into the broader European context. He was influenced to do this due to the already integrated European perspective of medicine which his book follows.Like now, medicine in 18th-century Europe was multicentric and characterised by rich intellectual activity, which contributed to the enhancement of clinical practice during this period. At the time, Greenland and Faroe Islands were also integrated into this European context because they contributed for medical-scientific development that would lay the foundations for modern medicine. Francesco Roncalli Parolino obtained just recognition for these regions through the advancement and defence of their valuable medical contributions.

SALOMONE ENRICO EMILIO FRANCO (1881-1950) AND 1938 ITALIAN RACIAL LAWS: FROM THE ADRIATIC TO THE MEDITERRANEAN SEA / SALOMONE ENRICO EMILIO FRANCO (1881-1950) DI FRONTE ALLE LEGGI RAZZIALI ITALIANE DEL 1938: DALL'ADRIATICO AL MEDITERRANEO

More than eighty years ago, the so-called Racial Laws banished Italian Jews from all their properties and places. The authors analyze the biography of Salomone Enrico Emilio Franco (1881-1950), a cosmopolite pathologist. Born in Trieste but raised in Venice, he had his medical degree in Padua and was a pathologist at the Venice Hospital, and then he went to Portugal. Franco founded the Institute of pathology of Lisbon University. He studied leishmaniosis and hematology. During WWI, he served as a volunteer in the Italian Army. He was then a full professor of pathology at the Universities of Sassari, Bari, and Pisa. However, he was obliged by the so-called Racial Laws to leave Italy and go to Palestine. He fought as a volunteer for the realization of the State of Israel and directed the Institute of Pathology in Jerusalem.

cyclic AMP Regulation and Its Command in the Pacemaker Channel HCN4.

Direct regulation of the pacemaker "funny" current (If) by cyclic AMP (cAMP) underlies heart rate modulation by the autonomic nervous system. At the molecular level, cAMP activates hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that drive If in sinoatrial node (SAN) myocytes. Even though HCN channel genes were identified more than 20 years ago, the understanding of how cAMP regulates their gating is still fragmented. Here we summarize present understanding on how the cAMP signal is transmitted from the cytosolic to the transmembrane (TM) domain in HCN4. We further discuss how detailed structural knowledge prompted the development of pharmacological/genetic tools for the control of cAMP regulation in these channels.

Rational design of a mutation to investigate the role of the brain protein TRIP8b in limiting the cAMP response of HCN channels in neurons.

TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) is the neuronal regulatory subunit of HCN channels, a family of voltage-dependent cation channels also modulated by direct cAMP binding. TRIP8b interacts with the C-terminal region of HCN channels and controls both channel trafficking and gating. The association of HCN channels with TRIP8b is required for the correct expression and subcellular targeting of the channel protein in vivo. TRIP8b controls HCN gating by interacting with the cyclic nucleotide-binding domain (CNBD) and competing for cAMP binding. Detailed structural knowledge of the complex between TRIP8b and CNBD was used as a starting point to engineer a mutant channel, whose gating is controlled by cAMP, but not by TRIP8b, while leaving TRIP8b-dependent regulation of channel trafficking unaltered. We found two-point mutations (N/A and C/D) in the loop connecting the CNBD to the C-linker (N-bundle loop) that, when combined, strongly reduce the binding of TRIP8b to CNBD, leaving cAMP affinity unaltered both in isolated CNBD and in the full-length protein. Proof-of-principle experiments performed in cultured cortical neurons confirm that the mutant channel provides a genetic tool for dissecting the two effects of TRIP8b (gating versus trafficking). This will allow the study of the functional role of the TRIP8b antagonism of cAMP binding, a thus far poorly investigated aspect of HCN physiology in neurons.

COVID 19 and Spanish flu pandemics: All it changes, nothing changes.

The Corona Virus 19 (COVID 19) epidemic is an infectious disease which was declared as a pandemic and hit all the Countries, all over the world, from the beginning of the year 2020. There are many similarities between the COVID 19 epidemic and the Spanish flu epidemic. We considered some preventive measures which do not change in the two epidemics.

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.

The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

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.