The anti-bacteria activities of MHACD were investigated against Staphylococcus aureus and Enterococcus species and the results showed that MHACD possesses a spectrum of activity against the two bacteria. The electrochemical cyclic voltammetry study on MHACD revealed that it is a redox active compound with promising catalytic properties in electrochemical applications. The inhibition potential of MHACD for mild steel corrosion in 1 M HCl was investigated using potentiodynamic polarization method. The results showed that MHACD inhibits steel corrosion as a mixed-type inhibitor, and the inhibition efficiency increases with increasing concentration of MHACD. The adsorption of MHACD obeys the Langmuir adsorption isotherm; it is spontaneous and involves competitive physisorption and chemisorption mechanisms. Quantum chemical calculations revealed that the energy of the highest occupied molecular orbital (HOMO) of MHACD is high enough to favor forward donation of charges to the metal during adsorption and corrosion inhibition. Natural bond orbital (NBO) analysis revealed the presence of various orbitals in the MHACD that are capable of donating or accepting electrons under favorable conditions.The macrocylic ligand, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (MHACD) was synthesized by the demetallation of its freshly synthesized Ni(II) complex (NiMHACD). Successful synthesis of NiMHACD and the free ligand (MHACD) was confirmed by various characterization techniques, including Fourier transform infra-red (FT-IR), proton nuclear magnetic resonance ( Macrocyclic ligands are important class of organic compounds that usually contain heteroatoms that are capable of chelating with substrates in their molecules . Most maFully saturated macrocyclic complexes consisting of six nitrogen atoms such as 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane are considerably less common ,29. The Staphylococcus aureus and Enterococcus species, while the anticorrosion potential was carried out on mild steel in 1 M HCl solution. The choice of 1 M HCl as the test solution for the corrosion inhibition study is premised on the frequent use of dilute hydrochloric acid in various industrial applications [This has motivated us to carry out the present study on the synthesis of MHACD via dematallation of its nickel(II) complex (NiMHACD). Successful demetallation was confirmed by various characterization methods including Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-vis) adsorption spectroscopy, and energy dispersion X-ray (EDX) techniques. In view of diverse applications of nitrogen-containing macrocyclic ligands as documented in literature ,25,26,27ications . It cons−1 for N–H bending vibrations, the broad band at 3550 cm−1 for N–H stretching and the band at the C–N band at 1026 cm−1. The 13C-NMR spectra  ion in a similar complex [The synthesis and characterization of NiMHACD and MHACD are summarized under the experimental section. The spectroscopic data to support successful synthesis of both NiMHACD and MHACD are listed in  spectra  shows thterature . The 1H- spectra  gave wel spectra  showed u spectra  revealed complex .−1 compared to that of NiMHACD by appearing at 3300 cm−1. The 13C-NMR of MHACD  and the concentration dependencies of the antibacterial activities of MHACD are shown in The MHACD was tested for possible activities against bacteria isolates from milk and groundwater samples. The Enterococcus species and the strength of antimicrobial activities generally increases with increasing concentration from 1 µg/mL to 4 µg/mL. The observed antimicrobial properties of MHACD may be attributed to the presence of N-donor groups in the molecule, which may serve as binding sites to favor the possible binding mechanism that leads to the deactivation of the microbes.The results showed that MHACD has a spectrum of activities against the selected microbes being more active against the −1 without and with 1 mM of MHACD are shown in −1) on the electrochemical behavior of MHACD was also investigated and reported in et al. [etc.The cyclic voltammogramms (CVs) obtained for Pt electrode in the phosphate buffer solution (PBS) at the scan rate (ν) of 25 mV·sn et al.  for thioDcc, the anodic peak potential, Epa was significantly linearly dependent on the log ν giving rise to the regression equation: The effect of scan rate on the electrocatalytic oxidation of MHACD at the Pt electrode as reported in p (peak current) versus log ν and Epversus log ν are shown in pvs. log ν  decreases with increasing concentration of the inhibitor, which is also an indication of the inhibitive effect of MHACD. The values of the percentage inhibition efficiency (%IEPDP) also revealed that the inhibition efficiency increases with increasing concentration.The reaction . In othe(sol)) and the water molecules on the metallic surface (H2O(ads)) according to the equation:x is the size ratio of the number of water molecules replaced by one molecule of organic substrate. Adsorption isotherms are often employed to gain more insights into the mechanism of corrosion inhibition, due to the fact that thermodynamic adsorption parameters provide information about the mode of interactions between the inhibitor molecules and the active sites on the metal surface [Kads is the equilibrium constant of the adsorption process and inhC is the concentration of the inhibitor. The Langmuir adsorption isotherm plot is shown in 2). The adsorption equilibrium constant, Kads, was obtained from the intercept and the change in Gibb’s free energy (∆Gads) for the adsorption process was obtained using the equation:The inhibition of metal corrosion by organic molecules is usually as a result of adsorption of the inhibitor molecules on the metal surface. Therefore, an insight into the inhibition mechanism can be gained from the adsorption isotherm. The adsorption of an organic adsorbate onto metal–solution interface can be represented by a substitutional adsorption process between the organic molecules in the aqueous solution phase  and lowest unoccupied molecular orbital (LUMO) electron density surfaces in etc. The bond lengths for the geminal C–H bonds (not shown) are also not equal. This indicates that the molecule is non-symmetric. The observed bond angles and dihedral angles (not shown) also revealed the non-planarity of the molecule. The HOMO graphical surface revealed that the HOMO orbitals are of sigma type as expected for the saturated molecule, and distributed over the C and N atoms in the ring and not extended to the C-atoms C1 and C39 of substituent methyl groups. The LUMO on the other hand is mainly found on the C1 and slightly extended to the vicinal C and N atoms. Some calculated quantum chemical parameters of MHACD are listed in HOMO and ELUMO of the synthesized ligand were compared with our recent reports on some macrocylic compounds such as porphyrins [et al. [This is reflected in the unequal values of the bond lengths found for bonds like C9–N5 and C35–N30; C6–N12 and C9–N14; rphyrins , phthalorphyrins . The HOMrphyrins , phthalorphyrins . This su [et al. , and as  [et al. ,39,46, a [et al. . The val2) has the highest tendency of electron acceptance from the d-orbitals of Fe followed by C25 (3dz2), then N38 (3dxz) and N30 (3dz2).To investigate the prospective orbitals in the molecule of MHACD that can donate and accept charges to/from the appropriate orbital of the metal, a full natural bond orbital (NBO) analysis was carried out. The abridged natural atomic orbital (NAO) occupancies are listed in 3OH), formaldehyde (CH2O), ethylenediamine (C2H8N2), methylamine (CH3NH2), acetonitrile (CH3CN), sodium cyanide, sodium hydroxide (NaOH) pellets, hydrochloric (HCl) acid, sodium dihydrogen orthophosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen orthophosphate (Na2HPO4), potassium ferrocyanide(II) (K4Fe(CN)6·3H2O), potassium hexacyano-ferrate(III) (C6FeK3N6) were commercially obtained from MERCK  and SIGMA-ALDRICH . Müller-Hinton Agar was purchased from Fluka . Microorganisms (Staphylococcus aureus and Enterococci) were obtained from the culture collection of the Department of Biological sciences, North-West University (Mafikeng Campus), Mmabatho, South Africa. Analytical reagent grade of disodium hydrogen orthophosphate, potassium ferrocyanide(II) and potassium hexacyano-ferrate(III) were utilized to prepare 0.1 mol·L−1 Phosphate buffer solution and 5 mM Fe(CN)63−/4− respectively.All chemicals and reagents were of analytical grade and were used without further purification. Methanol  complex (NiMHACD) was carried out as reported in the literature . Nickel −1): 1026s (C–N); 1503m ; 3222m , 3550b  13CNMR : three distinct peaks: 39.90 (–N–CH3), 71.20 (–N–C–N–), 172.60 (–N–CH2–CH2–N)FTIR : 0.9 , 1.2 , 1.5 , 7.2 1A1g→1A2g), 324 nm (1A1g→1B1g)UV-vis (nm): 214 (EDX: 22.5 % NiThe synthetic route is summarized in the schematic diagram as shown in Aqueous solutions (10 mL each) of the initially synthesized nickel (II) complex, NiL (5.64 g), sodium hydroxide pellets (6 g) and sodium cyanide (10 g) were mixed together and evaporated in a rotary evaporator until excess liquid was removed. The resulting crystalline solid was washed with acetonitrile and excess chloroform, and finally evaporated to dryness. The white crystalline solid was collected and air-dried to obtain MHACD as the final product.−1): 1421s (C–N); 1584m ; 3300b FTIR : three distinct peaks: 28.56 (–N–CH3), 78.43 (–N–C–N–), 174.60 1HNMR : 0.9 , 1.3 , 1.6 , 7.3 UV-vis (nm): 286 (n→σ*)EDX: 0 % NiThe synthetic route of MHACD is summarized in the schematic diagram as shown in −1 while the UV-vis electronic absorption spectra were recorded in the wavelength range of 200–400 nm. Energy Dispersive X-ray (EDX) spectra were carried out with EDX-720 obtained from Shimadzu . The proton (1H) and carbon-13 (13C) NMR spectra of the compounds were recorded on a Varian Gemini 300 Broadband NMR spectrometer at 300 MHz in CDCl3.Spectroscopic analyses were carried out on both the synthesized complex, NiMHACD and the free ligand, MHACD. FTIR spectrophotometer  and the UV-vis spectrophotometer  were used for the FTIR and UV-vis studies respectively. The infrared spectra were recorded between 700 and 4000 cm−3 M solution of the synthesized ligand prepared in 0.1 M phosphate buffer solution (PBS). A 5 mM Fe(CN)63−/4− solution was used as the redox probe. The effect of scan rates on the electrochemical behavior of MHACD was observed between 25 and 300 mV·s−1. Electrochemical studies were carried out on Metrohm Autolab potentiostat/galvanostat PGSTAT 302N supplied by Eco Chemie . The equipment is driven by the general purpose electrochemical system (GPES) software version 4.9. A conventional three-electrode electrochemical cell system was used with Ag|AgCl (in 3 M KCl), platinum wire and platinum rod as the reference, counter and working electrodes, respectively.Electron transport properties of MHACD were investigated by electrochemical experiment using the cyclic voltammetry (CV) technique. The CV profile was recorded for a 10Staphylococcus aureus and Enterococcus species isolated from milk and groundwater samples in the North West Province, South Africa. The identities of the isolates were confirmed using standard preliminary and confirmatory microbiology tests. The isolates were obtained from the Molecular Microbiology Laboratory, Department of Biological Sciences, North-West University, Mafikeng Campus and used for the antimicrobial assay. Different concentrations of the ligand, namely 1 µg/mL, 2 µg/mL, 3 µg/mL and 4 µg/mL, were prepared with sterile distilled. Sterilized paper discs of uniform diameter (6 mm) were cut and sterilized by autoclaving. The paper discs were soaked in the standard solutions of the ligand for 10 min. Bacterial suspensions of Staphylococcus aureus and Enterococci were prepared and aliquots of 100 µL from each suspension were spread-plated on Muller Hinton agar. The paper discs were aseptically placed on inoculated plates and incubated at 37 °C for 24 h. Isolates were also tested against ligands while a disc with no ligand concentration was included in each inoculated plate as a negative control. The diameters of the zone of inhibition of discs were recorded for different concentrations of the ligand.The antibacterial activity of MHACD was studied against 2. Prior to each corrosion rate measurement, MS samples were abraded with different grades of emery papers (600 to 1200 grits), washed thoroughly with distilled water, degreased with acetone, rinsed with distilled water and finally air-dried.Mild steel (MS) specimens of the composition (wt %) 0.02 P, 0.37 Mn, 0.03 S, 0.01 Mo, 0.039 Ni, 0.21 C and balance Fe were cut into a square dimension of 1 cm × 1 cm and embedded in epoxy resin exposing a surface are of 1 cmThe solution of the synthesized MHACD used as corrosion inhibitor was prepared in the concentration range of 10 ppm to 1000 ppm.−1. The linear Tafel segments of the anodic and cathodic polarization curves were extrapolated to the corrosion potential Ecorr to obtain electrochemical kinetic parameters including the corrosion current density (corri), anodic (aβ) and cathodic (cβ) Tafel slopes. The percentage inhibition efficiency was calculated by using the equation: Electrochemical corrosion measurements of MS in 1 M HCl without and with various concentrations of the synthesized ligand as inhibitor were conducted using the potentiodynamic polarization method. The conventional three-electrode electrochemical cell comprising the MS with freshly polished surface as the working electrode (WE), Ag|AgCl, 3 M KCl as the reference electrode (RE) and platinum rod as the counter electrode (CE) was used for all the measurements. The electrochemical cell was connected to the Metrohm Autolab PGSTAT 302N supplied by Eco Chemie . The MS was immersed in the aggressive solution for 30 min to ensure a stable open circuit potential (OCP) before each electrochemical measurement. The potentiodynamic polarization study was carried out by sweeping the MS electrode potential between −800 and −200 mV at a scan rate of 1 mV·sThe electrochemical corrosion measurements were conducted under unstirred conditions at 303 K.HOMO) and the energy of the lowest unoccupied molecular orbital (ELUMO) were calculated. The global electronegativity, χ (χ = −½(EHOMO + ELUMO)) and hardness, η (η = −½(ELUMO − EHOMO)) were also calculated. The fraction of electrons transferred (∆N) from the inhibitor molecule (donor) to the metallic (Fe) atom (acceptor) was calculated using the equation: [Quantum chemical calculations were carried out to gain further insight into the correlation between the molecular structure of the synthesized ligand and its corrosion inhibition properties. The structure of MHACD was modeled with the GaussView 5.0 software to obtain the starting geometry after which geometry optimization was carried out using the Windows based Gaussian 09 suite software (version D.01) . Full gequation: ,58.χ and η denote the electronegativity and hardness, respectively. A value of 7 eV/mol was used for ty scale . The nat1HNMR, 13CNMR, UV-Vis, and EDX techniques. The free ligand, MHACD, was investigated for its antimicrobial activities, electrochemical redox activities and corrosion inhibition potentials. The following conclusions can be drawn from the results: 1)1HNMR, 13CNMR, UV-Vis, and EDX characterization revealed successful synthesis of NiMHACD and dematallation to MHACD.The results FT-IR, 2)Staphylococcus aureus and Enterococcus species, being more active against the latter and the zone of inhibition increases with increasing concentration of MHACD.MHACD was found to exhibit anti-bacteria activities against 3)MHACD displayed electrochemical redox properties that suggest its possible catalytic properties in electrochemical applications.4)Potentiodynamic polarization study suggested that MHACD is a mixed-type corrosion inhibitor for mild steel in 1 M HCl.5)The adsorption of MHACD was found to be spontaneous, obey the Langmuir adsorption isotherm, and involve competitive physisorption and chemisorption mechanisms.6)Quantum chemical study showed that the HOMO of MHACD is high enough to favor forward donation of charges to the metal, while various orbitals in the MHACD that are capable of donating or accepting electrons were identified using the NBO analysis.The nickel(II) complex of 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (NiMHACD) was synthesized and later demetallated to obtain the corresponding free macrocylic ligand MHACD. Both NiMHACD and MHACD were characterized using the FT-IR,


The importance of diet in the pathophysiology of metabolic syndrome is well acknowledged ,2,3 and This Special Issue of Nutrients includes both evidence-based original research and state-of-the-art reviews and meta-analyses of the scientific literature. There are articles investigating different dietary regimens ,13,14,15This collection of papers shows that the selection of foods should be based on scientific evidence, knowing the properties of each dietary component.


Solomon was collecting evidence with a non-standard, non-epidemiological method for informed decision making. The Case of Re A  likewise demonstrates the complexity of bioethical decision-making , which became the binding ethical rule of the utmost importance is not in the Hippocratic Oath. It comes from The History of Epidemics, which is part of the Hippocratic corpus .The origins of bioethics, self-defined as “science of survival”, are diverse . They can-making . The Hipc corpus . The samFrom Hammurabi's Code to the moment when the German theologian Fritz Jahr published articles using the German term “Bio-Ethik” in 1927  there weThe first and most important stimulus for establishing evidence-based bioethics was fostered by a series of important events in research and clinical medicine: the Harvard Definition of Brain Death , the RoeBritish Medical Journal introducing the notion of evidence-based policies (The first to use the term “evidence-based medicine” was David M. Eddy in his work on population-level policies. He was also the first to link clinical practice guidelines and insurance coverage of new technologies with the idea of evidence-based bioethics. Two associations, the American College of Physicians and the American Heart Association, followed the initiative immediately in 1987 by publishing evidence-based guidelines for cardiovascular disease prevention. Another important step toward establishing evidence based medicine was made in the United Kingdom by Richard Smith's editorial in the policies . Finallypolicies .A parallel history of evidence-based bioethics started in 1979, when Tom Beauchamp and James Childress published Principles of Biomedical Ethics , connectHow do we define and measure good and bad ethical outcomes in medicine and health care? How to even measure the bioethics of happiness? Pragmatic ethics attempts to use philosophical methods to identify the morally correct course of action concerned with legal issues in the life sciences. Ethical pragmatists, such as John Dewey, thought that norms, principles, and moral criteria were likely to be improved as a result of philosophical inquiry. Henry Sidgwick introduced the idea of motive or intent in morality, and Peter Singer conceptualized the idea of preference into moral decision making.utility does not so clearly point to the ideas of pleasure and pain as the words happiness, and felicity do: nor does it lead us to the consideration of the number, of the interests affected ” (The idea of human happiness is a good example of the utilitarian theory approach to bioethics. The “greatest happiness principle” or the principle of utility, forms the cornerstone of Bentham's thought. Bentham tried to develop an operational concept for the scientific approach to human happiness by proposing a technical instrument “Felicific Calculus.” By “happiness,” he understood a predominance of “pleasure” over “pain.” In the Principles of Morals and Legislation he wrote: “The word fected ” . Benthamfected ” . The Galfected ” . Psycholfected ” .Empirical research in bioethics started at the end of the 20th century and was mostly influenced by developments in biological and clinical research, using methods from epidemiology and medical statistics. Writing during the first decade of the HIV/AIDS epidemic, Benjamin Freedman noted that “perception, rather than reality, controls the generation and resolution of ethical issues” . FreedmaAfter two decades of development, there is a long list of studies in favor of evidence-based bioethics. However, the scope of scientific evidence behind bioethics should not be limited exclusively to medical outcomes. As Goldenberg claimed “The qualitative, ethnographic, and phenomenological methods typically undertaken in empirical ethics are ranked low on the evidence-based hierarchy of knowledge…” . It is a


Wandering spleen, also referred to as ‘ptotic spleen’, is a rare clinical condition characterized by splenic migration form its normal left hypochondrial position to any other abdominal or pelvic position. Among the multifactorial etiologies proposed, laxity of the spleen’s primary supporting ligaments is the most agreed-upon hypothesis. We present one rare case of wandering spleen in an 11-year-old girl who presented with recurrent abdominal pain with no localizing features. Her abdominal examination revealed an intra-abdominal left iliac fossa lump with restricted mobility, which was confirmed as a wandering spleen by abdominal sonography and contrast-enhanced computed tomography. Intraoperatively, an infarcted spleen was encountered with tortuous, elongated, torsional splenic pedicle and a single dense adhesive band with descending colon. Splenectomy was offered to the patient. Post-operatively, the patient is healthy and symptom free at one-year follow-up. The rare clinical diagnosis of this condition, particularly in the paediatric age-group, makes it an enigma for the surgical world. Wandering spleen (WS), is a rare clinical condition, with only about 500 cases reported worldwide and an incidence rate of 0.2% ,2. One oAn 11-year-old girl presented to us with repeated episodes of intermittent, moderate-to-severe intensity, non-radiating pain in the right iliac fossa for the last six months. She had no history of fever, vomiting or urinary symptoms. On physical examination, a 10 x 4 cm intra-abdominal, ballotable, smooth-surfaced lump, which had restricted mobility with respiration, was palpated in the left iliac fossa. Her routine haematological and biochemical investigations were within normal limits. Abdominal sonography, and colour Doppler flow imaging revealed a 13 x 15 cm spleen with heterogeneous echogenicity, situated antero-inferior to the left kidney in the left iliac fossa with tortuous, elongated splenic vessels with torsion and a low blood-flow profile. Contrast-enhanced CT of the abdomen revealed a 15 x 17 cm spleen in the left iliac fossa, with a long, tortuous pedicle (approximately 15 cm) with torsion and focal areas of splenic parenchymal ischemia  that are directly associated with it  and two ligaments (pancreaticocolic and phrenicocolic) in indirect association. Failure of fusion of the dorsal mesogastrium to the posterior abdominal wall during embryogenesis leads to failure or defective attachment of these ligaments, leading to WS. The gastrosplenic, splenorenal and phrenicocolic ligaments have been primarily implicated (A second school of thought incriminates the hormonal changes and abdominal laxity in multiparous women as an acquired cause of WS and better explains the presence of WS in women of child-bearing age . Huge, hThe abnormal fixation of the spleen predisposes the splenic vascular pedicle to become tortuous, elongated and prone to intermittent torsion, in turn making the spleen vulnerable to infarction .Often labelled as a rare clinical diagnosis, especially in the paediatric population, the presentations of WS can be vivid . The speClinically, a mobile mass can be felt on abdominal examination. However, in our case the presence of a dense band between the spleen and descending colon restricted the mobility of the spleen. A sonographic examination of the abdomen usually suffices to delineate the location, size and any architectural deformities of the spleen in most cases. When the splenic vascularity is in question, either colour Doppler flow imaging or contrast-enhanced CT can both confirm the diagnosis and provide additional information on the blood flow profile in the splenic pedicle. Splenic parenchymal ischemia is characterized by a change in blood flow and a heterogeneous echogenicity of the spleen. This information plays a vital role in the pre-operative decision to offer the patient splenoplexy or splenectomy as the choice of surgery.The surgical intervention is defined by the vascularity of the spleen. A patient with splenic infarction due to torsion of the splenic pedicle, as in our case, is offered splenectomy. Splenoplexy, either open or laparoscopic, is offered to most of the other patients in whom splenic pedicle detorsion and splenic fixation to either the diaphragm or abdominal wall are done . NonoperWS is a rare condition that often presents as a clinical enigma. A clinician should have a high degree of suspicion for WS, particularly in women of child-bearing age and children who present with recurrent abdominal pain and a mobile abdominal mass. Modern imaging techniques are usually diagnostic and can identify the splenic pedicle torsion with a high degree of accuracy. Surgical intervention, in the form of either splenoplexy or splenectomy, is largely governed by the findings of pedicle torsion and the associated risk for acute splenic infarction.Conflict of interest statement: none declared.


Background: Though clinical guidelines for assessment and treatment of chronic subjective tinnitus do exist, a comprehensive review of those guidelines has not been performed. The objective of this review was to identify current clinical guidelines, and compare their recommendations for the assessment and treatment of subjective tinnitus in adults.Method: We systematically searched a range of sources for clinical guidelines  for the assessment and/or treatment of subjective tinnitus in adults. No restrictions on language or year of publication were applied to guidelines.Results: Clinical guidelines from Denmark, Germany, Sweden, The Netherlands, and the United States were included in the review. There was a high level of consistency across the guidelines with regard to recommendations for audiometric assessment, physical examination, use of a validated questionnaire(s) to assess tinnitus related distress, and referral to a psychologist when required. Cognitive behavioral treatment for tinnitus related distress, use of hearing aids in instances of hearing loss and recommendations against the use of medicines were consistent across the included guidelines. Differences between the guidelines centered on the use of imaging in assessment procedures and sound therapy as a form of treatment for tinnitus distress respectively.Conclusion: Given the level of commonality across tinnitus guidelines from different countries the development of a European guideline for the assessment and treatment of subjective tinnitus in adults seems feasible. This guideline would have the potential to benefit the large number of clinicians in countries where clinical guidelines do not yet exist, and would support standardization of treatment for patients across Europe. Tinnitus is essentially made up of two components, the phantom perception of a sound in the ears or head, and the degree of emotional reaction to that percept. Tinnitus can co-occur with several medical-otological disorders such as presbycusis, though etiology is unknown for the majority of tinnitus patients . This review was registered with PROSPERO, the international register of systematic reviews (protocol number: CRD42016038588) prior to commencing the literature search. The review was exempt from human ethics procedures as there were no human participants and only secondary sources of data  were used.The aims, the work plan, and the protocol for this systematic review were developed by TINNET Working Group 1, a COST Action BM1306 (2014–2018) to create a pan-European tinnitus research network . Those records were required to identify or describe themselves as guidelines, and be the most recent guideline form the country of origin. No publication date or language restrictions were imposed on the eligibility of the guidelines.Guidelines were excluded if they were for objective tinnitus, pediatrics, referred only to the triage or referral pathways for assessing and treating tinnitus, or if they were a guide for only one specific type of assessment or treatment procedure for tinnitus.www.guideline.gov), National Institute for Health and Clinical Excellence , Guideline International Network , Google, and hand-search of reference lists of any included guidelines was undertaken. International experts were also contacted to ask if they were aware of any guidelines that had not already been identified from the search results. The date that the search for guidelines was first conducted was 2 May 2016 and was undertaken by TEF and HH using “tinnitus” and “guideline” as the two key terms. The final search was conducted on 24 June 2016.The literature search for clinical guidelines included the Medline, PubMed, and the Cumulative Index to Nursing and Allied Health Literature (CINAHL), and EMBASE databases. In addition to these the National Guideline Clearinghouse  acted as an arbiter. As an additional check and in line with other systemic review searches using internet search engines, a Data extraction was undertaken using a tailored form that had been pilot tested and was emailed to reviewers in the form of an Excel spreadsheet. A document with guidance on the extraction of information for each of the items was provided to each of the reviewers to improve consistency of data extraction. Data extraction from each guideline was undertaken by at least two reviewers who were native speakers of or fluent in the language in which the guideline was published. Reviewers extracted information from the guidelines regarding items about the: country and year of publication, availability, author details, sponsor/funder involved, scope, target audience, developers and process related to the guideline, recommendations for assessment and treatment procedures, the level of evidence and type of rating system used  related to the recommendations, and items related to the implementation and revision of the guideline.HH and TEF were responsible for data management and maintained editorial rights. All identified records were saved into a Microsoft word master file and then saved in pdf-copy.www.agreetrust.org). It contains 23 items grouped under six guideline domains. Each item is scored on a 1–7 scale where 1 = “Strongly disagree” and 7 = “Strongly agree.” Scores are standardized to provide an overall percentage score. Previous reviews have used a 60% marker to distinguish high and low quality guidelines  all were developed during the last 5 years.Table Table 1Summary of guideline development by country.All the guidelines included information on the professional backgrounds of the participants in the respective development groups and in three out of the five cases , provided information on how views of funding bodies and competing interests were addressed. Although patient groups and the public were consulted in the development of three guidelines , the actual expected users of the guidelines were health professionals.Details were provided in all guidelines  about how literature was located and used to inform the respective recommendations. That is, details of search strategies using MeSH and other search terms and databases such as Medline and PsychInfo were included. Tools and criteria used to assess the evidence included the: Oxford Centre for Evidence Based Medicine (U.S. and German guidelines) and American Academy of Paediatrics' (American guideline) evidence criteria respectively, the AMSTAR checklist (Dutch guideline), and the GRADE ranking system of trust in conclusions of the literature (Dutch guideline). American, Danish, Dutch and German guidelines all provided information and referred to the research literature associated with each recommendation as well as describing their methods for reaching consensus on each recommendation. The Dutch, German and U.S. guidelines consider the strengths and limitations of the research literature and were reviewed externally prior to publication. Similarly, those three guidelines also state a year by and/or describe conditions under which they would be revised.Table All guidelines describe the need for physical examination by an ENT doctor, although physical examination is not explicitly referred to in the Swedish guideline. The American guideline recommends examination to exclude objective tinnitus, cardiovascular disease and vascular lesions, neurologic diseases, middle or outer ear infection/disease, vertigo, head-neck masses, or other treatable conditions. The German guideline additionally mentions cervical, dental, and temporomandibular joint functional exploration in a silent environment to evaluate tinnitus modulation.Audiological assessment was recommended in all the included guidelines. The majority refers to audiometry as a general category, but the German guideline provides most detail. For example, it specifies details relating to the assessment of oto-acoustic measurements, brainstem auditory evoked responses, caloric tests, determination of tinnitus loudness and frequency using narrow-band noise and pure tones, residual inhibition, Feldmann masking curves  to a psychologist or psychiatrist.The Tinnitus Handicap Inventory  as an assessment of tinnitus, The American and the Dutch guideline recommend against it, unless patients have one or more of: tinnitus that localizes to one ear, pulsatile tinnitus, focal neurological abnormalities, or asymmetric hearing loss. The German guideline also recommends against acoustic examination using sound pressure levels more than 84 dB 1 week after acute tinnitus or tinnitus exacerbation.Conduct a thorough physical examination to exclude possible  causes of tinnitus .Complete a thorough audiological assessment .Establish the degree to which a patient experiences subjective tinnitus as bothersome or distressing using a validated and reliable multi-item questionnaire such as the TQ, THI, TFI, or HADS .In situations where patients appear to be experiencing a degree of distress or difficulties related to living with tinnitus, consider making a referral for an assessment by a psychologist or psychiatrist .Variation exist in recommendations regarding the use of imaging studies .Table There is a consensus that medicines should not be prescribed for the treatment of subjective tinnitus, though some variation in the level of specificity that each guideline has. For example, the German guideline lists specific medicines that should not be prescribed for the treatment of tinnitus. The German and Swedish do however note that medicines such as antidepressants might be prescribed to treat comorbid conditions. Herbal supplements such as Gingko biloba are also specifically recommended against being used in all guidelines except for Sweden which does not make recommendations for or against their use.The use of hearing aids is recommended by all guidelines but only when clinically meaningful hearing loss is also present in people suffering from tinnitus. The use of a cochlear implant is mentioned in the Dutch and German guidelines and only recommended when there is profound hearing loss or deafness in addition to tinnitus. The Dutch guideline is the only one to provide scores on tinnitus questionnaires  for when such interventions should be considered .Psychological interventions for tinnitus can potentially include a wide range of components but there is general consensus on the use of two of them. In particular, the provision of information and education about tinnitus and treatment options is consistently recommended across the guidelines although there is some variation in the specificity of the content that each provides. Second, specialized CBT for tinnitus is specifically recommended by all the guidelines except for Sweden which mentions it only in relation to the presence of stress, anxiety, or depression.Least consistency exists across the guidelines in relation to TRT. Specifically, the Dutch guideline recommends that TRT can only be contemplated if tinnitus is very mild (TQ <30) and the patients specifically asks for TRT, the American guideline indicates that sound therapies “may” be recommended to patients with tinnitus, while the Swedish guideline recommends that sound stimulation be used as part of TRT for people without hearing loss. The German guideline recommends the use of notched music therapy, but recommends against the use of TRT.In relation to other less commonly used treatments (such as acupuncture or hyperbaric oxygen), the guidelines mostly indicate that there is an insufficient body of evidence to be able to make recommendations for or against their use.Lastly, three guidelines  either caution that there is insufficient evidence, or make additional recommendations against the use of transcranial magnetic stimulation (rTMS), transcranial direct current stimulation (tDCS), dietary supplements, neuromodulation treatments, and hearing aids for tinnitus patients without hearing loss.Provide information about tinnitus and treatment options .Use hearing aids only when patients also experience hearing loss .Specialised CBT for tinnitus should be offered to patients .There is a lack of consensus on the use of TRT for tinnitus.Prescribed medicines and herbal supplements should not be used for the treatment of tinnitus .Treatment with TMS is recommended against by Dutch and U.S. guidelines, and German guidelines give an “uncertain” recommendation.The AGREE II tool was used by the authors who undertook data extraction of the respective guidelines and the summarized results are shown in Table This systematic review aims to compare existing clinical guidelines for the assessment and treatment of subjective tinnitus in adults. Five guidelines, developed in the last 10 years within Europe, Scandinavia, and North America were included in the review. Although there are differences in some specific recommendations for assessment and treatment procedures across the guidelines, in general, commonalities across guidelines were high. The fact that there are differences in some of the recommendations is not surprising and appears to reflect the relatively young state of the field and the evolving nature of assessment and treatments for subjective tinnitus—a symptom with a high level of heterogeneity. On the other hand, the level of agreement, for example, in the recommendation of specialized cognitive behavioral therapy reflects the growing evidence base for the effectiveness of this treatment to alleviate patients' distress and impairment, even though significant changes in the tinnitus percept itself as a result of CBT have been proposed, though not yet assessed across studies.When the methods of the development of guidelines were reported, it was clear that the respective groups were making efforts to be transparent, systematic, and using the best available evidence base, and frequently linking recommendations to specific research literature. For example, systematic reviews and meta-analyses were referred to whenever available to inform recommendations. It should be noted though that there is a lack of high quality studies or powered randomized trials of some treatments either for practical or methodological reasons. Regardless, the strengths and limitations of the evidence for particular recommendations were included for the majority of the guidelines and thus enable the user/reader to make informed decisions about following the recommended actions. Furthermore, target users were generally clearly defined and the development groups were comprised of a range of the health professionals often involved in the assessment and treatment of tinnitus. These two factors are important not only for providing expert input into the guideline, but also for garnering “buy-in” from potential users of the guidelines and focussing the content.Differences in recommended assessment procedures tend to relate to specific techniques  rather than general principles , or the presence or absence of severe physical pathology that might be causing the tinnitus. Differences related to, for example, the recommended questionnaires for assessing tinnitus related interference and distress. While all the guidelines referred to the THI (the German guideline indirectly refers to this), only the American, Dutch and German guidelines referred to the TQ. Recommendations for specific questionnaires to measure psychological distress  also varied with some guidelines not mentioning any  and others such as the Dutch and Swedish guidelines which referred to the HADS. Differences also existed between the recommendations to assess loudness discomfort levels with the American and Dutch guidelines not recommending the use of such tests while the other guidelines did.With regard to treatments, differences are found primarily regarding recommendations for the use of sound therapies. TRT specifically is not recommended by the German guideline, conditionally by the Dutch guideline and the American guideline indicates that clinicians “may” recommend it; TRT is currently being tested in a large multicenter trial in the U.S. . A lack of evidences about other treatments such as acupuncture, hyperbaric oxygen and some herbal supplements leads most groups to recommend against them. The American guideline though is more cautious and simply states that because there is a lack of evidence they can neither recommend for or against the use of such treatments.Differences in the recommendations of assessment and treatment procedures could be explained by a combination of factors including the time of the development of the guideline and availability of translated versions of the questionnaires , the known psychometric properties of the questionnaires themselves [e.g., concerns have been raised about the cross-cultural use of the HADS  in a way (such as stepped care) that is minimally burdensome to the patient. That is patients who are assessed as having relatively little tinnitus related distress and interference should receive less intensive treatment in the first instance, than someone who is assessed as having severe levels of distress and interference in activities of daily living.There are two critical factors that affect the conclusions that can be drawn from the included guidelines. Firstly, and as with all systematic reviews, the search strategy and inclusion criteria used determine what is located and subsequently included. In this review, we used the search terms “tinnitus” and “guideline” to conduct the search in a wide range of databases, repositories of clinical guidelines, and search engines, with the intention of being focussed enough to identify the most relevant documents within a manageable number of search results. Only including the term “guideline” though might have resulted in relevant documents, albeit not called “guidelines,” being omitted from search results. Similarly, our use of inclusion/exclusion criteria that led to the decision to exclude documents such as the TRI flowchart could be problematic as it (the TRI flowchart) is a comprehensive document potentially used in many situations to inform assessment and treatment decisions.To minimize the risk of omitting relevant search results we contacted a range of international experts and members of guideline development groups. In addition to this, we conducted hand searches of the references lists of included guidelines for relevant sources. We also recruited native speakers to extract data from the respective guidelines in an effort to ensure that data collection was as accurate as possible. It is possible, that different search and inclusion criteria might have led to different documents being included. However, given the large range of assessment and treatment options and the limited evidence base around many treatments in particular, it is unlikely that our conclusions would differ significantly if further guidelines had been identified at this time. Future systematic reviews though will be able to use this as a reference point.As researchers from around the world are collecting and making efforts to better understand the heterogeneity of subjective tinnitus in adults and systematically evaluate assessment and treatment options, we have, for the first time, described the major similarities and differences between existing clinical guidelines for subjective tinnitus in adults. The results reveal true guidelines from only five countries and thus highlight a need to develop guidelines that are endorsed by the range of professionals involved in assessing and treating tinnitus. Although we do not place a great deal of weight on the quality assessment ratings of the guidelines, they do suggest that there is room for improvement particularly with regard to implementation and evaluation. The absence of guidelines contributes to the variations that exist in assessment and treatment of tinnitus internationally.Clinical.” As with existing clinical guidelines, attention will need to be given to how the future European guideline is disseminated, subsequently evaluated, and the implications for resource management considered. We expect it to be challenging task but one that will hopefully result in a more reliable and equitable assessment and treatment of tinnitus patients across Europe.While it would be tempting to do so, it is beyond the scope of this paper to formulate a new or composite guideline based on the results that we have obtained. Instead, the results of this review in conjunction with those from a survey of European tinnitus healthcare providers and researchers  supported the collaboration between the authors and the formation of the COST Action BM1306 (2014–2018) TINNET Working Group I.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.


Over two billion people suffer from micronutrient deficiencies. Food fortification is a prominent nutrition intervention to combat such deficiencies; however, its effectiveness, risks, and ethical implications vary depending on the contexts associated with the deficiency it is addressing and the circumstances with its implementation. The aim of this research was to analyse the profile of nutrition interventions for combating micronutrient deficiency with particular focus on food fortification reported in existing systematic reviews (SRs), guidelines and policy statements, and implementation actions for nutrition. A review of secondary data available from online databases of SRs, guidelines and policy statements, and implementation actions, categorised as either “nutrition-specific interventions” (NSpI) or “nutrition-sensitive interventions” (NSeI), was conducted. Currently, there is evidence available for a diversity of food fortification topics, and there has been much translation into action. Indeed, food fortification and micronutrient supplementation interventions and NSpI more broadly dominate the profile of interventions for which there were SRs, guidelines, and policy statements available. The findings demonstrate that, although there is a rational linear relationship between evidence synthesis and translation in formulating policy and actions to combat micronutrient deficiencies, the various nutrition interventions available to help combat micronutrient deficiencies are not equally represented in the evidence synthesis and translation processes. Effective and safe policies and actions to combat micronutrient deficiencies require decisions to be informed from a body of evidence that consists of evidence from a variety of interventions. Into the future, investment in making available a higher number of SRs, guidelines and policy statements, and actions of NSeI is indicated. From 19 to 21 November 2014, the Rome Declaration on Nutrition, the outcome document of the Second International Conference on Nutrition (ICN2), noted that over two billion people suffer from micronutrient deficiencies and in particular vitamin A, iodine, iron, and zinc  have rec(i)Nutrition-specific interventions (NSpI) “address the immediate causes of undernutrition, like inadequate dietary intake, and some of the underlying causes, like feeding practices and access to food” . Food fo(ii)Nutrition-sensitive interventions (NSeI) “can address some of the underlying and basic causes of malnutrition by incorporating nutrition goals and actions from a wide range of sectors. They can also serve as delivery platforms for nutrition-specific interventions” . InterveHowever, food fortification is just one among many nutrition interventions available to combat micronutrient deficiencies. These interventions are commonly considered as belonging to either of two broad categories:Whether or not to select food fortification as an intervention to combat a micronutrient deficiency will depend largely on the circumstances associated with this nutrition problem and what alternative interventions are available. In the short term, a NSpI such as food fortification may be well suited to treat an acute micronutrient deficiency. Alternatively, in a chronic situation, food-based NSeI are likely to be better suited to protect against and sustainably prevent micronutrient deficiencies. Over the longer term, food- and diet-based interventions are better able to tackle the co-existence of multiple micronutrient deficiencies, and deliver social and economic co-benefits . GeneralEvidence-informed policy and practice decisions are required to plan and implement nutrition policies and interventions. The WHO “Handbook for Guideline Development”  explainsThree forms of evidence synthesis and translation were analysed: Published SRs; nutrition guidelines and policy statements; and implementation of nutrition actions.We searched and selected records for inclusion as follows:To identify relevant SRs and SR protocols, we conducted a rapid search of four databases, namely PubMed, Epistemonikos, TRIP (31 May 2016), and Health Evidence (3 June 2016). The Cochrane Database of Systematic Reviews (CDSR) had been previously screened for another study (31 July 2015) to identify nutrition-related reviews. We searched this database for reviews that met the inclusion criteria for this study. The search strategies employed for the first four listed databases are outlined in Eligible studies were SRs and SR protocols of interventions that addressed any primary micronutrient deficiencies, either through fortification (including biofortification and targeted fortification) or other methods, in any population. Systematic reviews of nutrition interventions that addressed outcomes regarded as well-established manifestations of micronutrient deficiencies such as poor growth and adverse neurodevelopmental outcomes were also included. Systematic reviews were defined as those reviews that had predetermined objectives, had predetermined criteria for eligibility, searched for at least two data sources of which one needed to be an electronic database, and performed data extraction and risk of bias assessment .“Category 1 interventions are interventions for which there are guidelines that have been recently approved by the WHO Guidelines Review Committee (GRC). Category 1 interventions also include those supported by recommendations and other forms of guidance that have been adopted or endorsed by the World Health Assembly”“Category 2 interventions are interventions for which systematic review(s) have been conducted but no recent guidelines are yet available that have been approved by the WHO Guidelines Review Committee”“Category 3 interventions are interventions for which available evidence is limited and systematic reviews have not yet been conducted”Data for nutrition guidelines and policy statements were collected from the WHO e-Library of Evidence for Nutrition Actions (eLENA) . This daTo identify relevant records, the complete list of interventions included in the eLENA was screened by title (and content if necessary) by one author (KW) and cross-checked by another author (ML). Disagreements were resolved through discussion. Interventions that sought to prevent or treat micronutrient deficiencies were eligible for inclusion. Interventions addressing macronutrient deficiencies were excluded, as were those addressing overweight/obesity or diet-related non-communicable diseases. Additionally excluded was mandatory folic acid fortification as an intervention to reduce the risk of neural tube defects (NTDs). The rationale for this exclusion was that the relationship between folic acid and NTD risk reduction is not conceptually equivalent to that associated with the causation of a conventional micronutrient deficiency disease. Although the precise mechanism by which folic acid exerts its protective influence is not known, NTD risk is reduced when a “dose” of folic acid, beyond that required to meet the folate recommended dietary intake level, is consumed by at-risk individuals, i.e., in this circumstance, folic acid is acting more as a therapeutic agent than as a conventional nutrient . InterveData on the implementation of nutrition actions were collected by interrogating the WHO Global database on the Implementation of Nutrition Action (GINA) . In thisWe categorized interventions addressed in the included reviews as fortification, supplementation, or other interventions. For those that addressed fortification, we extracted information on whether fortification was with single micronutrients  or with multiple micronutrients , as well as the type of food vehicle used. We calculated the proportions of included SRs and SR protocols assessing food fortification, supplementation, and other types of interventions for micronutrient deficiencies, as well as proportions of reviews addressing food fortification with single or multiple micronutrients. Data were presented descriptively.We classified included interventions by intervention type  and by the level of evidence supporting them . We then calculated the number and proportion of interventions by type and by category. Interventions were also classified as NSpI or NSeI and described accordingly.Sub-topics that met the inclusion criteria were classified by the type of nutrition actions being implemented . The number and proportion of nutrition actions were then calculated. Actions were also classified as NSpI or NSeI and described accordingly.The review selection process for SRs and SR protocols is outlined in n = 29, 17.0%) or micronutrient supplementation-only interventions   .n = 5, iodine n = 4, zinc n = 3, vitamin A n = 1, vitamin D n = 1, and calcium and vitamin D n = 1). Vehicles for fortification included staple foods, maize flour, wheat flour, rice, salt, condiments, seasonings and various foods , as well as dairy and non-dairy beverages. The vast majority of the supplementation-only interventions involved single nutrients (n = 92) rather than multiple micronutrients (n = 16).Of the 29 fortification-only interventions, 14 involved fortification with multiple micronutrients and 15 involved single micronutrients  or a combined intervention . Other interventions included food-based interventions , targeted financial incentives , agricultural interventions , and education-based interventions. Combined interventions included fortification and supplementation (n = 6), fortification, supplementation and other interventions (n = 7), supplementation and other interventions (n = 3), and fortification and other interventions (n = 5).Some SRs and SR protocols addressed other interventions . Of the fortification interventions, four of seven were Category 1 or 2 interventions, and three of seven were Category 3 interventions. A number of other relevant interventions were also identified. Other interventions included those addressing feeding practices, marketing of infant formula, nutrition assessment, nutrition education, sanitation, and financial incentives. All other interventions were classified as either Category 1 or Category 2 interventions (n = 28).All interventions involving supplementation were classified as Category 1 or 2 interventions  were NSpI. The remaining six interventions (8.7%) were NSeI . Five of the six NSeI interventions were classified as Category 1 or Category 2 interventions.Of the 69 included interventions, the vast majority  or micronutrient supplementation , but more than half did not . Vehicles for food fortification included rice, maize or corn flour, wheat flour, milk, condiments and seasonings, sugar, margarine, butter, and oil. Some actions involved multiple micronutrients, such as fortification of salt with iron, iodine, vitamin B12, folic acid, zinc, and vitamin A. A number of nutrition actions involved the addition of multiple micronutrient powders to foods in the home. Nutrition actions involving micronutrient supplementation involved single nutrients  or multiple micronutrients. Other nutrition actions included both NSpI and NSeI. Overall, the majority of actions were NSpI . The remaining 477 actions were NSeI .Some nutrition actions involved food fortification   and the It was beyond the scope of this research to investigate specific explanations for the observed dominance of NSpI in general, and supplementation and fortification interventions in particular, among the interventions available to combat micronutrient deficiencies. However, the literature does provide examples where methodological orthodoxies and political factors can influence decision-making and result in the privileging of supplementation and food fortification interventions over NSeI. Methodologically, evidence synthesis orthodoxies typically rate the evidence obtained from studies using reductionist methods higher than evidence obtained from studies using more holistic methods. Investigations of relationships between single nutrients and single physiological outcomes are more amenable to reductionist methods than the inherently complex relationships that exist between NSeI and health outcomes ,27. PoliThe strengths of this research included the novel nature of the investigation, the high quality databases from which the data were sourced and the critical analysis of the collective profile of these data, their inter-relationships, potential knowledge gaps, implications for public health nutrition practice, and future research priorities. Nonetheless, the potential for limitations with the collection and analysis of the data cannot be discounted. Comprehensive data on systematic reviews were collected from five key databases; however, it is possible that we missed relevant SRs. Similarly, we may have missed relevant nutrition guidelines and policy statements and actions that are being implemented but are not recorded in the eLENA and the GINA, respectively. In the GINA the data extraction was restricted to actions only and not policies. It is possible that, if policies had also been included, the findings may have differed to a certain extent. The decision not to include policies was based on the authors’ experience that the existence of a policy does not always lead to an action, and it was action that was the priority interest for this investigation. Additionally, in relation to the GINA, the search relied on the classifications in the website database rather than on original keyword search, so relevant results may have been missed. Despite quality assurance procedures being undertaken, it is possible there were inaccuracies regarding data extraction, coding, and interpretation during the data analysis component of the research. We did not assess the quality of individual reviews, guidelines, or actions included in the study, as this would have been beyond the scope of the research aim. We also did not investigate the reach and impact of individual interventions. Therefore, a food fortification intervention to modify the nutrient composition of the national food supply and an NSeI such as a local agriculture program were recorded as one intervention each, despite the reality that the former would have a significantly higher reach and impact.To the authors’ knowledge this is the first study to investigate SRs, guidelines and policy statements, and actions by type of nutrition intervention to combat micronutrient deficiencies, as well as analyse the relationship between these three activities. A number of unanswered questions remain. Priority areas for future research include investigating the relationship between evidence synthesized for NSeI and what then is happening in policy and practice, especially if this can contribute to the informing of strategic combinations of interventions to combat micronutrient deficiencies; extending the investigation to analyse policy documents in the GINA; and investigating how and why there is a dominance of NSpI over NSeI in the observed SRs, guidelines and policy statements, and actions observed.Food fortification can be a powerful intervention for helping combat micronutrient deficiencies, especially when planned and implemented as a component of a strategic combination of interventions. Currently, there is evidence available for a range of food fortification interventions, and there has been much translation into action. However, food fortification and especially micronutrient supplementation interventions are dominating what evidence is being synthesised, which may subsequently determine what guidelines and policy statements are being produced. Effective and safe policies and actions to combat micronutrient deficiencies will require decisions to be informed by a body of evidence on a variety of interventions. This does not necessarily mean less investment in NSpI but rather more investment in prioritizing and proactively conducting and making available SRs of alternative interventions that go beyond linear relationships between nutrients and health outcomes, and extending to mid-stream and upstream interventions, such as those addressing socio-ecological and environmental determinants. An increased variety in the types of interventions for which evidence synthesis is available would help inform a greater diversity of guidelines and policy statements and actions to counter the current dominance of supplementation and fortification interventions and increase the likelihood of comprehensive, sustainable, effective, and safe policies and actions to combat micronutrient deficiencies.


Huddling behaviour in neonatal rodents reduces the metabolic costs of physiological thermoregulation. However, animals continue to huddle into adulthood, at ambient temperatures where they are able to sustain a basal metabolism in isolation from the huddle. This ‘filial huddling’ in older animals is known to be guided by olfactory rather than thermal cues. The present study aimed to test whether thermally rewarding contacts between young mice, experienced when thermogenesis in brown adipose fat tissue (BAT) is highest, could give rise to olfactory preferences that persist as filial huddling interactions in adults. To this end, a simple model was constructed to fit existing data on the development of mouse thermal physiology and behaviour. The form of the model that emerged yields a remarkable explanation for filial huddling; associative learning maintains huddling into adulthood via processes that reduce thermodynamic entropy from BAT metabolism and increase information about social ordering among littermates. For example, honeybee swarms cluster to form structures whose surface density varies with the environment temperature, maintaining the porous core at thermal homeostasis . Common The huddling behaviour of laboratory rodents, in particular rats and mice, has emerged as a model system for the study of social thermoregulation. This is due to their prevalence as laboratory species, and to the precision with which the thermal physiology of these species has been investigated ,13. A nuThe individual behaviour from which rodent huddling emerges has been described formally as ‘homeothermotaxis’; turning in the direction that brings the body temperature closer to a preferred temperature ,19. As sCentral to thermodynamic descriptions of huddling is a role for brown adipose fat tissue (BAT), which evolved as the organ of thermogenesis in early mammals ,24, and As precocial mammals develop, maximal energy in BAT stores at birth decay, as does the drive towards huddling based on physiological demands ,26,39,40react to thermal stimuli, to help minimize the energetic costs of physiological thermoregulation, have since been supplanted by more elaborate  circuitry that enables animals to predict the thermal consequences of contact with conspecifics [Emerging theories of social thermoregulation in primates are beginning to extrapolate from the data on rodent huddling to explain how complex human psychological concepts, such as the establishment of distinct attachment styles, and the formation of internal models for social behaviour, may be supported by the same neural systems that regulate huddling behaviours in rats and mice , such aspecifics . The idepecifics , who proTo make explicit these assumptions about the role of odour-heat conditioning in social thermoregulation, and to test the idea that social thermoregulation can provide a scaffold for the development of non-thermal social behaviours, the current study presents a model, based on the thermodynamic description of rodent huddling by Wilson , which p2.A Monte Carlo algorithm has recently been shown to capture the statistics of huddling behaviour , i.e. tha, then selecting a second pup at random from a different group, b, and then either joining together the groups to which pups a and b belong with probability ρa,b, or detaching a from its group to form a new group of size 1, whereT is referred to as the ‘temperature parameter’. Predicting how huddling statistics change over developmental time thus requires a definition of how T, which determines the likelihood of two groups joining to form a larger huddle, changes as the thermal physiology matures.More precisely, the huddling algorithm involves iteratively selecting a pup at random, and the development of thermal behaviour in the same animals and under the same experimental conditions. The basis of the model is therefore a dataset published by Eedy & Ogilvie [Although studies of the developing thermal physiology of mice are numerous, few have quantified the relationship between the maturing thermal physiology  Ogilvie , which dT, develops. To investigate the transition from physiological to filial huddling, the data of [T; then values of T were fed through the huddling model  to predn=7 pups joined in a single group, and then evaluates equation  and final (p0) measurements to define p′=(p−p0)/(p1−p0), the rescaled points p′ were well approximated by an exponential decay,k a time constant describing the rate of depletion of BAT reserves.The points gerspetz . Using tb shows the mass-specific metabolic rate  reported by Eedy & Ogilvie [G, defined asS is the entropy associated with P. As such, k is a unit of energy, and the fit obtained using k=8.31 ascribes particular significance to k as the universal gas constant [Figure 1 Ogilvie , and a fconstant . Thus thEntropy is a measure of disorder, describing the number of different configurations of a system that are possible. Only in open systems can entropy decrease, i.e. in systems that exchange matter with the surrounding environment. From day 8, 3.2.a shows that the growth curves measured by Eedy & Ogilvie [c=19 is a constant  and T1 is a temperature.The growth curves of mice typically display an initial rise (of decreasing magnitude) followed by the more familiar sigmoidal shape that is often modelled as a logistic/autocatalytic function  on a thermocline, as recorded by Eedy & Ogilvie , are shofigure 2Tp+T1 or Tp−T2, suggesting that thermoregulatory systems based on BAT energy metabolism (responsible for T1) and total (non-muscle) metabolism (responsible for T2) are in competition for behavioural thermoregulation. Compared with the dominance of BAT in determining the preferred temperature in the first few postnatal days, the later dominance of other thermoregulatory systems represented by T2 may correspond with increases in e.g. liver, thyroid and adrenal activity [On each postnatal day, the animals appear to select either activity .3.3.T1 and T2 into a prediction of how the mean group size varies with age as the physiology matures from ectothermy to endothermy, using T∝T1−T2. Figure 3a (light trace) confirms that huddling (average group size) is predicted to decrease as the preferred temperature falls.The Monte Carlo method developed by Wilson  equatio) can be α for the odour of each of its littermates. The thermodynamic temperature parameter T is then weighted at each time step by the strength of association of pup b for the odour of pup a,β=0.2 is an arbitrary scaling constant. Note that in the control condition, where huddling interactions are determined only by the maturing physiology, α=1 for all strengths of association.To model how pups learn from huddling interactions, each is assumed to maintain a strength of association a and b as a conditioning trial. On each iteration of the huddling algorithm, the strength of association of pup a for the odour of pup b, αa,b, is updated to better predict whether future contacts between them will be maintained,r≡1 whenever the groups of a and b are combined, or r≡0 otherwise, and the learning rate is set to γ=0.001.The Delta rule from animal learning theory  can thena (heavy trace) shows that when littermates can learn to predict the outcome of huddling interactions, large huddles persist into adulthood, with larger huddles predicted from around day 15 in littermates able to learn compared to non-learning controls. Figure 3b shows the associative strengths learnt by an arbitrary pup, which grow to an early peak for all littermates, but thereafter increasingly discriminate among the littermates. The development of two associative strengths are highlighted, showing how littermates can learn strong positive or negative associations to one another. This emergent heterogeneity of preferences for maintaining contacts with specific littermates may constitute the basis of a rudimentary social system.Figure 34.P=et/k−, as is the typical profile for a precocial mammal [M=k(1−P). As such, the unit of energy k may be interpreted as a conversion factor between the (mass-specific) energy stored in fat tissue and the (absolute) muscle mass. The energy released in the conversion between fat and muscle is reflected by a spike in the entropy of BAT, measured as a peak in the mass-specific metabolic rate, i.e. the oxygen consumption. This spike is similar to the fluctuation in entropy (or heat capacity) measured by physicists during a Schottky anomaly , or , or P=e−aly  and predictive behaviour . More detailed models of associative learning are expected to yield qualitatively similar results.The results of adding odour-heat conditioning to the model of  providesa priori prediction that odour-heat conditioning might lead to the persistence of huddling into adulthood, the additional prediction that associative strengths among the litter can develop to be increasingly positive or negative was not anticipated, although the effect is simple to understand post hoc. Once two pups have developed a moderate association, an early contact that by chance results in a surprising outcome will lead to an adjustment in association that makes that predicted outcome less likely to result from future encounters. For example, two pups that expect to maintain contact (α>0) but do not, will adjust α to predict that future contacts are unlikely to be maintained (Δα<0), and in doing so reduce the probability of maintaining future contacts. Steady increases or steady decreases in associative strength between pups (b) thus reflect an amplification of early individual differences between them and result from closing the loop between behaviour .Beyond the een pups b thus reequation ) and leaequation ). By driequation  towards These dynamics demonstrate the usefulness of computational modelling as a tool for teasing out the predictions of how even simple systems behave when learning is allowed to interact with behaviour. The new prediction in this case is that while associative learning enables huddling to persist into adulthood, individuals will become increasingly selective in their choice of huddling partners. This prediction could in principle be tested by measuring the changing preferences of animals, at thermoneutral temperatures, to huddle with surrogate objects scented with different littermate odours in a forced-choice experimental set-up. In similar set-ups using artificial odours, rats have been shown to spend more time on postnatal day 6 exploring odours recently paired with the experience of warmth, whereas on days 7 and 8 rats spend less time exploring an odour paired with the experience of cold, compared to baseline times spent exploring the warmer of two odour-neutral locations .a intersect defines a transition from physiological to filial huddling, i.e. at around postnatal day 15.Filial huddling preferences can be induced by exposure to novel odours when paired with soft contacts and/or warm environments (rather than food ). The effigure 3r), suggesting that a combination of the thermal and tactile properties of a contact determine its valence as a reward for odour conditioning. The emergence of filial huddling shown in figure 3a is thus consistent with the general finding that preferences for conspecifics are stronger for older (15–20 days) animals than younger animals (5–10 days), and are disrupted in older animals to a greater extent by blocking olfaction  [Day 15 represents a landmark in the development of rat social thermoregulation. Preferences measured in rats on day 15 can be induced by a single 2 h exposure at postnatal day 14 to a scented object other than the mother, only if it is both warm and soft (consistent with the definition of ulphate) . An impoAccording to the model, mechanisms of classical conditioning, which presumably involve the pre-optic region of the hypothalamus ,46,60–62