MfERG-RADs were similar in both groups. NPDR eyes showed a significant (p < 0.05) linear correlation between RADs and both, CC FDa and FD%. The EZ “normalized” reflectivity was negatively correlated with CC FD% in NoDR eyes. In NPDR T1D eyes a significant relationship between abnormal outer retina functional responses and CC impairment was observed, while in NoDR eyes the photoreceptor reflectivity was correlated to CC abnormalities. The outer retina dysfunction in NPDR correlated to CC drop-out let hypothesize that the outer retinal elements are functionally impaired in proportion to the CC vascular supply deficit.To study the outer retina morpho-functional characteristics and the choriocapillaris (CC) features in type 1 diabetic (T1D) patients, with and without signs of diabetic retinopathy (NPDR and NoDR). Twenty-five NPDR and 18 NoDR eyes were imaged by Optical Coherence Tomography Angiography. Ellipsoid zone (EZ) “normalized” reflectivity and CC perfusion density parameters, as flow deficits number (FDn), flow deficit average area (FDa) and flow deficit percentage (FD%), were analysed. Multifocal electroretinogram (mfERG) response amplitude densities (RADs) were measured. Mean EZ “normalized” reflectivity, CC FDn and FD% values, were similar ( By using the optical coherence tomography angiography (OCTA) it was possible to detect the non-perfusion areas at different retinal layers, in particular at the level of deep capillary plexus (DCP) that nourishes the middle and outer retina3.More recently, the choriocapillaris (CC) and choroid (Ch) have been recognized by means of optical coherence tomography (OCT)/OCTA as key-players in the diabetic pathological mechanism, and the name of “diabetic choroidopathy” was coined to describe the CC hypoperfusion as a relevant and early physio-pathological process in diabetic patients7. The photoreceptor structural integrity can be quantitatively investigated measuring the ellipsoid zone (EZ) reflectivity, a novel standardized methodology that was used to quantify photoreceptors’ damage in different disorders, including type 2 diabetic retinopathy (DR)8.The CC and Ch sustain the high metabolic rate of the outer retinal layers and retinal pigment epithelium, contributing to the photoreceptor oxygen supply10, supporting the concept of early neuro-retinal dysfunction before retinal vascular signs of diabetic retinopathy are visible. However, due to the novel concept of diabetic choroidopathy3, it would be of a great interest to understand whether there exists an influence of CC and Ch changes on outer retinal function in T1D patients.In T1D patients, multifocal electroretinogram (mfERG) abnormalities have been reportedTherefore, the aim of our study was to study the outer retina morpho-functional characteristics and the choriocapillaris features in T1D patients with and without signs of diabetic retinopathy, by using OCTA and mfERG, for investigating on the potential influence of CC and Ch changes on outer retinal function.p = 0.064).Forty-three eyes of 43 T1D patients  were included; 25 with signs of non-proliferative diabetic retinopathy (NPDR group)  and 18 without signs of diabetic retinopathy (NoDR group) . Of the 25 NPDR group, 14 eyes had mild and 11 moderate NPDR. Demographic results were showed in Table No statistically significant differences have been found between NoDR and NPDR groups in EZ “normalized” reflectivity, CC Flow Deficit number (FDn), CC Flow Deficit percentage (FD%).p = 0.018) (Table The mean ± SD CC Flow Deficit average area (FDa) was statistically significant greater in NPDR group 37.12 ± 14.17) compared with NoDR group (27.82 ± 7.55) , while no correlation was found in NPDR group. All the other relationship showed no statistically significance.The EZ “normalized” reflectivity was negatively correlated with CC FD% in NoDR (p = 0.02) and FD% (p = 0.007), respectively; no significant correlation was found between age and both, EZ “normalized” reflectivity and FDn. In NPDR group the diabetes duration and HbA1c were not correlated with EZ “normalized” reflectivity , CC FDn , CC FDa  and FD% .We correlated the EZ “normalized” reflectivity, the CC FDn, the CC FDa and the FD% with age, diabetes duration and glycated hemoglobin (HbA1c) levels. In NPDR group we found a significant relationship between age and CC FDa (p = 0.011), while no other correlation was found with EZ “normalized” reflectivity (p = 0.135), FDn (p = 0.377) and FD% (p = 0.088); the diabetes duration was significantly correlated with both, CC FDa (p = 0.009) and FD% (p = 0.027), while no correlation was found with EZ “normalized” reflectivity (p = 0.082) and FDn (p = 0.637); none of these parameters was correlated with HbA1c levels .In NoDR group the age was significantly correlated with CC FDa (p > 0.05) differences were found between NoDR and NPDR eyes  recorded from isolated rings R1, R2, R3) and combined rings (R1 + R2 and R1 + R2 + R3), not statistically significant  correlations between mean RADs and choriocapillaris features  in NoDR group. By contrast, NPDR eyes showed a significant (p < 0.05) linear correlation between mean RADs from R3 and R1 + R2 + R3 and both CC FDa and CC FD% from almost superimposable areas (Table p = 0.075). No other significant correlations between RADs and CC characteristics were found in other examined areas.We found no significant linear (p > 0.05).Mean mfERG RADs from all rings plotted as a function of age, diabetes duration and HbA1c level were not significantly linearly correlated in NoDR group . In these relationships, the correlation line had a negative slope thus meaning that reduced parafoveal RADs were correlated with increasing age in NPDR eyes. No other significant correlations between electrophysiological data and diabetes duration and HbA1c% were detected.In NPDR group we found a significant linear correlation between age and mfERG RADs from R2, R3 and R1 + R2  in T1D eyes without DR. More importantly, we showed a significant relationship between abnormal outer retina functional responses and CC features (CC FDa and CC FD%) in NPDR T1D eyes 14, and another recent interesting study showed microvascular changes in the retinal (SCP and DCP) and choroidal levels (CC) in both, T1D and T2D patients, compared to controls, even in the absence of DR (NoDR)15.Several previous studies already reported a CC hypoperfusion in diabetic eyes12, and we reported worsening of CC flow between NoDR and NPDR groups.Our data agree and support this evidence: in fact, as already demonstrated, the reduction of CC flow increases with the advancing of diabetic retinopathyWhen correlating the EZ “normalized” reflectivity with CC FD parameters, we found a negative correlation between EZ “normalized” reflectivity and CC FD% in NoDR group.13.Dai et al., suggested that the CC FD might represent an earlier preclinical marker of microvascular dysfunction, as demonstrated by the significantly increased in CC FD in NoDR eyes compared to controls16, confirming the involvement of choroid in diabetic physio-pathological events (“diabetic choroidopathy”)18. Indeed, photoreceptors and CC have been described as elements of a symbiotic unit in which dysfunction of their components are strictly correlated also in a different degenerative pathology, like intermediate age-related macular degeneration (iAMD)19.This result could support the hypothesis that the CC vascular dropout is associated with photoreceptors’ damage also before the development of diabetic retinopathyBy contrast, not statistically significant relationship has been found between EZ “normalized” reflectivity and CC FD parameters in TD1 NPDR group.9 by applying both ring and sector analyses to signals recorded from the macular area up to 10° of foveal eccentricity. As already described9, although outer retinal signals are different between controls and T1D eyes, our data confirms that mfERG is not a tool able to discriminate between different stages of the DR disorder.When comparing electro-functional data between NoDR and NPDR eyes, we did not find any significant differences in all studied areas, meaning that the bioelectrical function of the pre-ganglionic elements is grossly similar either in absence or presence of minimal clinical signs of diabetic retinopathy. This agrees with all that found in our previous studyMoreover, when correlating functional data with OCTA parameters, we found a significant correlation between reduced mfERG RADs and increased CC FDa and CC FD% in R3 and the combined R1 + R2 + R3 areas in NPDR eyes.This finding suggests that only in presence of clinical evident signs of DR the photoreceptors and bipolar cells’ dysfunction correlates with the CC flow impairment, and significantly only in the parafoveal areas or in the combined foveal and parafoveal areas.4.This means that for NPDR eyes, in those localized areas of the macula where a major number of retinal abnormalities are visible , the underneath outer retinal elements are functionally impaired in proportion to choroidal vascular supply deficitThe above-mentioned correlation with CC features is not present in NoDR eyes with absence of microvascular retinal abnormalities and with good glycaemic control. It is likely that in the early stage of DR, the impairment of the CC flow is not associated to the precocious parafoveal retinal dysfunction.In addition, we did not find either in NPDR or in NoDR eyes any significant correlation between mfERG data and EZ “normalized” reflectivity. This finding may be ascribed to the fact that when recording data by mfERG we mainly select the functional activity of photoreceptors and bipolar cells and their synapse, which mainly involves the outer nuclear and outer plexiform layers. It is likely that the precocious reflectivity impairment of the EZ can eventually interfere with the activation of phototransduction occurring at the level of the outer segment of photoreceptors and not with the neuronal signalling impairment involving outer and middle retinal layers, thus it could not be captured by the mfERG responses. Another hypothesis could be that, in the diabetic neurovascular disorder, the choroidal ischemia and the CC flow deficit are likely able to induce a precocious preganglionic element dysfunction, before the photoreceptors’ structural damage is evident.20.In our study, exploring the correlation between CC parameters and age, we found a significant correlation between CC FDa and CC FD% with age in NPDR and CC FDa and age in NoDR group. These correlations were expected and were in line with a recent study that showed a strong negative correlation between CC FD and ageing in healthy subjects in foveal, parafoveal and perifoveal areas12; in fact, even if in NoDR group no ophthalmoscopic sign of DR were observed, the CC perfusion could be affected by a diabetes longer time disease. These correlations could have lost in NPDR groups, in which diabetes duration may be a part of a greater complex pathological mechanism.Interestingly, we also found a correlation between diabetes duration and CC FDa and CC FD% in NoDR group. These correlations could represent another aspect of the pathological event series for which CC flow impairment worsens with advancing diabetic disease9. This correlation could not be confirmed in the very early stages of the disease, as also the overall glycaemic load and disease duration were not correlated with retinal dysfunction in all examined areas in both groups. Our sample consisted of well controlled metabolic patients and this aspect could be responsible of the absence of a significant correlation between retinal morpho-functional dysfunction and HbA1c.As for the relationship between mfERG RAD values and descriptive demographics in NoDR and NPDR examined patients, we found a significant correlation between reduced preganglionic bioelectrical activity and increasing age in the parafoveal areas only in NPDR eyes. This finding confirms the already known concept of significant functional deterioration of photoreceptors and bipolar cells in older T1D patients, when signs of diabetic lesions are visibleOur study presents some limitations, which includes its cross-sectional design and the relatively small sample size. Future prospective longitudinal studies on the retinal and choroidal perfusion in diabetic eyes should help to better understand the relationship between CC perfusion and photoreceptor damage.In conclusion in this study we aimed to explore the relationship between outer retina morphology and function and choriocapillaris changes in type 1 diabetic patients with and without signs of diabetic retinopathy. Interestingly we found in NPDR T1D eyes a significant relationship between abnormal outer retina functional responses and CC impairment and in T1D eyes without DR a significant relationship between EZ “normalized” reflectivity and CC features.This observational cross-sectional study enrolled T1D eyes with non-proliferative diabetic retinopathy (NPDR) and T1D without DR (NoDR) at the Department of Ophthalmology, IRCCS-Fondazione Bietti, Rome, was approved by the Institutional Review Board of IRCCS—Fondazione Bietti, Rome (protocol n. Ret03/2016) and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants.21.Two experienced examiners (MP and EC) identified the eyes with NPDR based on the analysis of color fundus photographs, according to the modified ETDRS retinopathy severity scale and analyzed the B-scan OCT images to exclude the presence of edemaPatients received a complete ophthalmologic examination, which included the measurement of BCVA using ETDRS visual charts, intraocular pressure (IOP), and dilated fundus examination.Exclusion criteria were: presence of macular edema as assessed by fundus examination and confirmed by OCT exam, previous ocular surgery or intravitreal injection therapy, any maculopathy caused by other than DR, significant lens opacity and refractive error > -6 diopters spherical equivalent (SE) or >  + 4 diopters SE. Poor quality images with a signal strength index (SSI) lower than 6 for the PLEX Elite Swept Source (SS)-OCTA or with significant motion artifacts (seen as large dark or grey lines on the enface angiograms) were also excluded.22.All patients underwent OCTA imaging using SS-OCTA PLEX Elite 9000 device  with a 3 × 3 field of view area centered on the foveaThe main outcome measures were: ellipsoid zone (EZ) “normalized” reflectivity and choriocapillaris (CC) perfusion density. The CC PD was analyzed as: CC flow deficits number (FDn), CC flow deficit average area (FDa) and CC flow deficit percentage (FD%).8.All measurements were performed in a circular region of interest (ROI) centered on the fovea and with a radius of 1.25 mm. This decision was made because of enface images limited lateral resolution8. Briefly, the EZ enface image was exported after checking for a correct segmentation and then imported into Fiji ImageJ software . The mean brightness of the EZ enface image was measured. Because the structures’ brightness may depend on a variety of uncontrollable factors, thus a previously described image-processing algorithm to normalize the signal across a cohort was used24.The EZ “normalized” reflectivity was investigated as previously reported25. The obtained CC images were imported in Fiji ImageJ  and each CC image was compensated adjusting for shadowing artifacts and removing retinal vessel projection artifacts26 and then binarized using the Phansalkar method (with a window radius of 3 pixels)28. In order to quantify the CC flow, the “analyze particles” command was employed. The CC flow was finally measured using the following variables: (i) the FD number (FDn) that corresponds to the number of flow voids within the circular ROI, (ii) the FD average area (FDa) that corresponds to the average size of the flow voids and (iii) the FD percentage (FD%) that corresponds to the total percentage of flow voids within the ROI.The CC enface OCTA images were segmented using a 20 µm thick slab, which was placed 29 µm under the RPE fit boundary, that corresponds to Bruch’s membrane29 and to our previously published method by the VERIS Clinic 4.9  system32.In NoDR and NPDR eyes, mfERG was recorded according to the ISCEV standards29.After automatic rejection of artifacts by the software, the first order kernel response was examined to measure the function of the pre-ganglionic retinal elements (photoreceptors and bipolar cells)2) between the first negative (N1) and the first positive (P1) peaks, obtained from three concentric annular retinal regions (rings = R) centered on the fovea. Ring analysis was used to differentiate changes of the bioelectrical responses of the central foveal region with respect to the more eccentric ones in the macular area. Therefore, we analyzed the N1-P1 RADs derived from 0 to 2.5 , from 2.5 to 5 , from 5 to 10 , from 0 to 5 (R1 + R2) and from 0 to 10 (R1 + R2 + R3) degrees. To compare mfERG and OCTA results from almost superimposable areas, we referred to photoreceptor topography as in Curcio et al.33.The functional main outcome measure was the averaged response amplitude density  was estimated following the methodology used in our previous workNoDR, NPDR OCTA and mfERG data followed a normal distribution based on Anderson–Darling and Kolmogorov Smirnov tests.We used a one-way analysis of variance (ANOVA) to test whether groups’ OCTA and mfERG parameters’ means differ. We focused our attention on OCTA EZ “normalized” reflectivity, FDn, FDa and FD % to compare groups’ means.We compared mfERG groups’ parameters: R1, R2, R3, R1 + R2, R1 + R2 + R3 N1-P1 RADs. Each mfERG parameter was linearly regressed with each OCTA parameter by means of Pearson test for NoDR and NPDR groups and pooled together. Clinical features  were linearly regressed with mfERG RADs and OCTA parameters for each group.OCTA EZ “normalized” reflectivity was linearly regressed with OCTA parameters: FDn, FDa and FD % for NoDR and NPDR groups. A p-value ≤ 0.05 was chosen as statistically significant.


A scaling theoryof statistical (Markov) polyampholytes is developedto understand how sequence correlations, that is, the blockiness ofpositive and negative charges, influences conformational behavior.An increase in the charge patchiness leads to stronger correlationattractions between oppositely charged monomers, but simultaneously,it creates a higher charge imbalance in the polyampholyte. A competitionbetween effective short-range attractions and long-range Coulomb repulsionsinduces globular, pearl-necklace, or fully stretched chain conformations,depending on the average length of the block of like charges. Thenecklace structure and the underlying distribution of the net chargeare also controlled by the sequence. Sufficiently long blocks allowfor charge migration from globular beads (pearls) to strings, therebyproviding a nonmonotonic change in the number of necklace beads asthe blockiness increases. The sequence-dependent structure of polyampholytenecklaces is confirmed by molecular dynamics simulations. The findingspresented here provide a framework for understanding the sequence-encodedconformations of synthetic polyampholytes and intrinsically disorderedproteins (IDPs). There is considerable interest in understanding theirconformational behaviors and their phase behavior in solution, whichis relevant in a wide range of contexts, from the formation of biologicalsubcellular compartments to the design of underwater adhesives.11 the macroscopicphase separation (or “self-coacervation”12) that arises in PA solutions represents a reasonablemodel with which to study their formation.14 It is now known that the extensive clustering of opposite chargesin the primary structure of IPDs facilitates the spontaneous self-assemblyof MOs in vivo and in vitro.15 The availableexperimental results have been theoretically rationalized, first16 within the random phase approximation (RPA)following the method of ref (19IDPs are a pervasive component of the membraneless organelles(MOs)that exist within living cells;20 Alternating PAs have zero net charge and, ifsufficiently long, form globules.23 However, PAs with an ideal random distribution of positively andnegatively charged monomers can adopt a necklace-like conformationdue to the statistical deviations of the global PA charge from theensemble-average zero value.28 Pearl-necklace conformations are often encountered in hydrophobicpolyelectrolytes (PEs). They were first predicted theoretically andin simulations31 and later observed in experiments.35 Necklace formation in PAs andhydrophobic PEs is the manifestation of the Rayleigh instability36 of the charged spherical  globule. The only difference is that, in PEs, the attractive interactionsthat facilitate globular conformations have a hydrophobic nature,whereas in PAs they are due to sequence-dependent Coulomb attractionsbetween opposite charges.Less is known about the effects of sequenceand charge distributionor statistics on the single-chain conformations of PAs and IDPs.38 Their continuous phosphorylationlasts many hours, until the increasing net charge leads to a conformationaltransition within them. The latter triggers a set of biochemical processesthat results in the reset of the circadian clocks.38In vivo, the mechanism that maintainscircadian rhythms is basedon the Rayleigh instability of special IDPs, which serve as a molecularhourglass.N monomers, each with size a. A fraction f of the monomers is ionic;they are spaced equidistantly and each carries a charge ±e. The quenched sequence of positive and negative chargesobeys first-order Markov process statistics with correlation parameter40pij = p(i|j) is the conditional probability to have an ionic monomerof type i after one of type j , and p++ = p–– maintainsthe statistical neutrality of the molecules. By setting λ =−1 and 0, one recovers alternating and ideally random statisticsof charges, respectively. The limiting case of λ = 1 correspondsto a stoichiometric mixture of polyanions and polycations.40 The average charge of the block of consecutivelike charges is 1 + Λ with41f ≃ Λ/f monomers, and eachPA consists of about e units) is4141 PAs are immersed in a salt-free Θ solventwith dielectric constant ϵ, and u = lB/a = e2/ϵakBT isits dimensionless Bjerrum length. A more detailed description of thePA model can be found elsewhere.41In this work, we develop a scaling theory of statisticallyneutralMarkov PAs and demonstrate that PA sequence controls the conformationsof the molecules, including necklace formation and structure. We considerflexible PAs containing Q = 0  pairwise interactions between the permanentdipoles is given by W/kBT ≃ −lB2p4/r6, and the correspondingsecond virial coefficient is given by Bdip ≃ −a2u2d ≃ −a3u2f–1/2. Dipole–dipole attractions are balancedby three-body repulsions between all PA monomers, Bdipnnip2 + Cn3 = 0, where C ≃ a6 is the third virialcoefficient; n and ndip ≃ fn are the concentrations of all monomersand dipoles. This yields the equilibrium polymer volume fraction (density)of the globule formed by alternating PA23a ≃ aϕa–1 ≃ au–2f–3/2.We startour analysis with globally neutral PAs, also ref ). Theyfg ≃ (ξ/a)2 being the number of monomers per blob. Theinterior of the PA globule is a correlated melt of oppositely chargedblobs: The closest neighbors of the positively charged blob predominantlyhave a negative charge and vice versa.2 The energy of Coulomb correlation attractions per blob equals Fattr/kBT ≃ lBq2/ξ, and it is balanced by three-body repulsions with the energy Frep/kBT ≃ ξ3Cn3 ≃1. The resulting blob size is given by ξr ≃ a/ufΛ andthe globule density reads23As Λ increasesand the charge statistics gradually changesfrom alternating to ideally random (Λ = 1), each concentrationblob ceases to be electrically neutral, as shown in 23The latter increases with increasing sequence correlationsas soonas Λ exceedsfgr ≃ fξr2/a2. The crossover between essentiallyrandom and highly blocky sequences is defined by23qe is thecharge of the electrostatic blob.44 At Λ > Λr/hb, the charge of the concentration blob is equal to q ≃ fg, and the balance betweenCoulomb attractions and short-range repulsions, Fattr ≃ Frep, resultsin the globule density23hb ≃ a(uf2)−1/3 ≃ ξe. In other words, forPA sequences with high charge blockiness, the globule interior issimilar to that of a polyelectrolyte complex coacervate,46 as seen in 47This picture of the globule formed by essentiallyrandom PA holdsuntil all charged monomers within the concentration blob are of thesame sign, that is, until the block charge Λ is on the orderof their number, Q ≠ 0. According to Rayleigh’scriterion,36 the spherical globule splitsinto the smaller globules (beads or pearls) connected by the stretchedchain fragments (strings) if the energy of repulsions between theexcess charges FCoulglob/kBT ≃ lBQ2/R exceeds the surface energy of the globule Fsurfglob. The characteristic global charge of statistically neutral MarkovPAs equals Q =0 for alternating sequences, Λ = 0, to Q ≃ fN for block lengths comparable to the chain length, Λ≃ fN.41 The globuleradius equals Rglob ≃ a(N/ϕ)1/3, and its density ϕis given by Fsurfglob/kBT ≃ R2/ξ2 with ξ ≃ a/ϕ.We are now in a position to consider theinstability of the sphericalPA globule with respect to the necklace formation induced by its nonzerocharge, I between the free energies that destabilizeand stabilize the spherical globulea/r, the global charge of the PAs is low and the spherical globule isstable; (ii) for random sequences, Λa/r ≪Λ ≪ Λr/hb, (some) globules split intoseveral beads; (iii) for highly blocky sequences, Λ ≫Λr/hb, the formation of a necklace consisting ofa large number of beads is expected.The ratio Below we consider the conformationsof Markov PAs over the entirerange of charge blockiness, Λ ≥ 0 , and delineate five conformational regimes, includingthree corresponding to pearl-necklaces of various types.a/r, PAstatistics are close to alternating, and the globalcharge is not sufficient to perturb the spherical globule of the radiusAt Λ ≪ Λa/r ≪ Λ ≪ Λr/hb, the global charge of the PA is close to the threshold value thattriggers necklace formation  and length on the charge statistics Λin Regime II is remarkable and stems from the exact compensation oftwo opposing tendencies. As the charge blockiness increases, Coulombcorrelation attractions get stronger, ϕr ∼Λ, thereby providing a higher surface tension of the globule/beadand a higher stability against splitting. Simultaneously, a decreasingglobule/bead size (I(Λ) ≃ 1 foundin The independence ofthe necklace structure −2/3 is the number of monomers in the electrostaticblob. The number of the necklace beadsAt Λ≫ Λ47Qbead/Dbead ≃ Qstr/lstr, one can find the bead chargeQstr ≃ Λ.We notethat, in Regime IV, a minor fraction of the necklace charge is stilllocated in the beads. Using the equality between the electrostaticpotentials of the bead and the string,f and the chain length N become comparable.At Λ≃ ΛPE, when the PAs comprise several chargeblocks, Nbead ≃ 1 and the necklace length Lnec ≃ au1/3f2/3N given by N+ ≠ N–).47 Each tadpole consists of an almostneutral globular head and an extended PE tail (or two tails) carryingalmost the entire net charge of the PA.47 At |N+ – N–| ≃ N+ ≃ N, the head size and charge found in ref  providing the dominant contributionto R, the number of globular beads Nbead changes in a nonmonotonic fashion and reaches a maximumat Λ ≃ Λb/s, as seen in The results of our scaling analysis aresummarized in N/ge)1/9, while otherquantitiesremain continuous. Our results predict ensemble-averaged propertiesof PAs, while the effect of the sequence realization, that is, quenched(frozen) disorder,53 on their conformation remains to be explored. Focused numericalsimulations25 covering the vicinity of thistransition would help clarify how sequence disorder smears it out,and affects the cooperativity expected from the Landau theorem.54 How quenched sequences under thermal agitationfall  in Regime III or IV and whether both structurescan follow each other along the same sequence of a reasonable lengthare of particular interest.In our analysis, we have primarily adopted a mean-fieldapproachthat neglects quenched charge fluctuations, except in regime IV, whereblockiness is essential. At the III/IV transition, the bead chargeis found to drop by a factor .The equalitybetween the block and the bead charges defines boundary III/IV andis accompanied by the migration of the non-neutralized charge fromnecklace beads to strings and by an Nbead decrease with increasing charge blockiness.In conclusion, the sequence specificity of Markov PA conformationshas been analyzed, and five conformational regimes have been delineated.The boundaries between these regimes are controlled by the interplaybetween the inherent sequence scales and the characteristic physicallengths of the problem.


Aspergillus flavus originated compounds that may act as drug leads to treat future infections.Penicillin was the first and most famous fungal secondary metabolite used as broad spectrum antibiotic that revolutionarised pharmaceutical research and also saved millions of lives. The over optimistic belief in 1967 that sufficient antibiotics had been discovered to defeat infectious diseases was quickly crashed with the appearance of multidrug resistant (MDR) bacteria in 1990s. This has posed a serious threat to mankind. Although scientists are making efforts to synthesize and discover new antibiotics there are not enough new drugs in pharmaceutical pipeline to beat the pace at which MDR bacteria are emerging. In view of this there is an urgent and serious medical need for new bioactive compounds to be discovered to treat infections caused by MDR pathogens. The present study is aimed to investigate the antibacterial potential of Mentha piperetta, one was processed for isolation of secondary metabolites on the basis of preliminary antibacterial testing. Observation of morphological and microscopic features helped in identification of the fungal strain as Aspergillus flavus. Potato Dextrose Agar (PDA) medium was used for fungal growth while Czapec Yeast Broth (CYB) medium was used for production of fungal metabolites. Column chromatography technique was utilized for purification of compound from crude fungal extract and the mass of the compound was determined using Liquid Chromatography Mass Spectrometry (LCMS) method. Structure elucidation of the pure compound was performed using 500 Varian Nuclear Magnetic Resonance (NMR) machine. Docking was performed using Glide SP algorithm. Agar well diffusion method was used to determine the invitro antibacterial potential of the compound against two MDR bacterial strains i.e. Staphylococcus aureus and Proteus vulgaris. For this a total of 4 dose concentrations i.e.  of the compound were prepared and applied to bacterial strains on Mueller Hinton agar using tetracycline as control.Among the 6 isolated fungal strains from the rhizosphere of A. flavus was determined as (2E)-3- prop-2-enoic acid with the formula C14H14O5 and exact mass of 262.08. The in-Silico analysis showed that this compound has the potential to inhibit the binding pocket of S. aureus TyrRS (1JII) with docking score of − 8.67 Kcal mole− 1. The results obtained from invitro experiments were encouraging as at 1000 μg mL− 1 the compound showed 58.8% inhibition against S. aureus and 28% inhibition against P. vulgaris.The chemical name of the purified compound from 14H14O5 and exact mass of 262 exhibited antibacterial potential both insilico and invitro against both Gram negative and Gram positive bacteria. The compound was more active against S. aureus in comparison to P. vulgaris. From the obtained results it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.The pure compound with formula C The emergence of multi-drug resistant pathogens results in serious and life-threatening human infections. This results in pressing demands for the entry of new antibiotics into the existing cohorts . BioactiAspergillus genus has the highest innovation index regarding the development of biologically active secondary metabolites H+ respectively, calculated for the compound with the formula C14H14O5 and exact mass of 262.08.Solvent system comprising of Ethyl acetate-13C NMR (Broad Band and DEPT) were used to establish the molecular formula of the known compound. HR–ESI–MS showed pseudo molecular ion peaks [M+] at m/z 262.1112 [M]H+ and 284.2121 [M]Na+ that correspond to molecular formula C14H14O5 [Calculated as C14H14O5 + Na = 284.2121]. The spectrum got from IR spectroscopy showed occurrence of hydroxyl group (3350 cm− 1), carbonyl group at (1740 cm− 1), and aromatic ring (1635 and 1381 cm− 1). The absorption maxima in the UV spectrum (320 nm) also showed the occurrence of an aromatic ring. The pure compound (262) at a concentration of 6 mg was mixed in 0.65 mL of deuterated chloroform and a series of 1-dimensional and 2-dimensional NMR analyses were performed on 500 MHz NMR Spectrometer.The HR–ESI–MS and δ 1.36 and δ 1.39 corresponded to two CH3 with an integration of three protons. The remaining signals represented the integration of single proton The two aromatic protons were signaled at δ 7.21 and δ 7.52 as doublets The two signals at chemical shift 6.5 ppm and 6.9 ppm (both doublets) revealed as alkenyl hydrogens attached to the carboxyl group.The Proton NMR represented total of eight proton signals. Two signals at 13C NMR gave 14 signals that corresponded to 14 carbons skeleton of the compound, exhibiting 4 quaternary carbons at δ 125.5, δ 161.3,δ 125.5 and δ 138.1while 2 carbonyl carbons at δ 170.2 and 168.9 respectively. The 2D-HSQC exhibited 8 protonated carbons as given in Table 1. The two dimensional (1H–1H COSY and HMBC) represented different correlations and the structure is elucidated as given in Fig. 1H–1H COSY, the methine proton of H–3 showed correlation with the proton of C–4, also with the methyl protons of C–12, while the H–4 showed correlation with the methyl protons of C–11. The proton H–5 showed correlation the proton of C–6, and the proton H–13 showed correlation the proton of C–14 and vice versa. While in HMBC correlations were compared with the compound already reported in literature. The 2D (1H–1H COSY and HMBC) alone with UV and IR were compared with the values in literature  prop-2-enoic acid. The structure of the compound and the values when searched online in dictionary of natural products showed similar mass and UV with that of a polyketide derivative reported from endophytic fungus Pestalotiopsis species  prop-2-enoic acid. Our results showed strong interaction with key amino acids residues of target proteins with a dock score of − 8.67 Kcal mole− 1  which states that compounds isolated from terrestrial Aspergillus species have the potential to inhibit MDR gram positive and gram negative bacterial strains [Aspergillus have been reported to have broad spectrum antibacterial activity among which some are currently used as commercial antibiotics [Aspergillus species a compound called CJ-17,665 showed activity against different MDR bacteria including multi-drug resistant S. aureus (MDRSA).For in-vitro screening of the compound, two MDR bacterial pathogens i.e.  strains . In the ibiotics –29. AmonThe purified compound and its inhibitory activity against MDR bacterial strains presented here projects the possibility of this compound as a new antibacterial drug. The compound has the ability to retard the growth of both gram positive and gram negative bacterial strains by showing a prominent zone of inhibition. The purified compound may be considered as potential antibacterial candidate to be used against the two test MDR bacterial strains. However, further studies are needed to confirm our findings and a number of selective toxicity assays needs to be perform before its application as a new antibacterial compound.This study has a few limitations. Primarily the amount of the purified compound was only 10 mg so as a result the sample size is very small. The compound was tested against only one gram positive and one gram negative bacterial strains. Secondly, due to limited amount of the compound minimum inhibitory concentrations (MICs) could not be performed. Selective toxicity of the compound was not tested. In future the study will be expanded by optimizing the culture conditions that will help to purify greater amount to the compound. Sample size will be increased by increasing the number of test bacterial strains. Selective toxicity of the compound will be tested using animal models which will not only help to enhance the impact but will also help in further refinement of this study.A. flavus has the potential to inhibit the growth of two test bacterial strains. In comparison to P. vulgaris the compound more actively inhibited the growth of S. aureus. Further, it is concluded that this compound can be used as potent antibacterial candidate but further studies will be needed prior to its use as antibiotic.The present study concludes from results of insilico and invitro experiments that the compound purified from


Takotsubo cardiomyopathy (TC) is a rare, reversible cause of left ventricular wall motion abnormality (LVWMA) that mimics the presentation of acute myocardial infarction (AMI). TC is usually preceded by an emotional or physical stressor and appears to be more common in postmenopausal women. Various pathophysiological hypotheses of TC have been proposed, but the exact mechanism of action remains elusive. Elevated levels of catecholamines leading to cardiac dysfunction are the most prevalent hypothesis. The protective role of estrogen in the development of cardiomyopathies has been studied extensively. International Takotsubo Diagnostic Criteria (InterTAK) and Mayo clinic diagnostic criteria both have the stipulation stating prevalence of TC is higher in postmenopausal women which hints towards the protective role of estrogen in the development of TC. To review the protective role of estrogen in the mechanism of this novel pathology, we searched Pubmed and Google scholar for the relevant articles by using keywords such as: “takotsubo cardiomyopathy”, “apical ballooning”, “broken heart syndrome”, “stress cardiomyopathy”, “left ventricle wall motion abnormality”, “estrogen”, “estradiol” and “sex hormones”. Our research revealed that although the prevalence of TC is greater in postmenopausal women as compared to men, the prognosis is worse in men. It also revealed the involvement of multiple cellular pathways under the influence of estrogen that could explain the cardioprotective effect of estrogen. Most of the articles found were based on animal studies, thus, there is an emphasis on future human studies. However, we strongly suggest evaluating estrogen levels as part of the initial workup for any patient presenting with signs and symptoms of cardiac pathology. EpidemiologyThe prevalence of takotsubo cardiomyopathy (TC) is 1%-2% in patients presenting with symptoms of acute coronary syndrome (ACS) . BecauseBackgroundTC is a rare disease that has recently started to gain recognition. TC was first described by Satoh 1990) in Japan who derived the name of the pathology from the shape of the container which is used to catch octopus  . The sha990 in JaThe exact pathophysiology of TC is unknown. The most widely accepted hypothesis is the elevated level of catecholamines is preceded by an emotional or physical trigger, as presented by Wittstein et al. (2005) . The stuNumerous diagnostic criteria have been proposed due to the novelty and complexity of the disease. In 2008, Mayo Clinic proposed a revised diagnostic criteria that had been widely used . Ghadri The predominance of TC in postmenopausal women suggests the protective role of estrogen. It has been well established by Ostadal et al. (2014) that the incidence of ischemic heart disease (IHD) is lower in premenopausal women as compared to postmenopausal women . Ostada This review article aims to further explore the cardioprotective effect of estrogen. We aimed to reveal the changes that occur at the cellular level under the influence of estrogen and the estrogen-mediated effects through three kinds of estrogen receptors such as estrogen receptor- alpha (ER-ɑ), estrogen receptor- beta (ER-β) and G-protein coupled estrogen receptor (GPER). In this review article, we searched Pubmed and Google scholar using the following keywords: “takotsubo cardiomyopathy”, “apical ballooning”, “stress cardiomyopathy”, “ broken-heart syndrome”, “cardiomyopathy”, “ estrogen”, “sex hormones” and “ estradiol”. Our search revealed 181 articles. After screening the title and abstract of each article from the year 2000 onwards, we included 20 articles that directly discussed the effects of estrogen and its receptors. In this review, we also discuss the impact of each type of ER individually and mention their protective effect against TC.Estrogen is a sex hormone produced by the ovaries and is responsible for the development of secondary sexual characteristics in females. It has been well established that premenopausal women have a lower incidence of cardiac events as compared to men . Also, tA breakthrough study to mimic the presentation of TC in vivo was done by Ueyama et al. (2007) . Their mTo understand the association of estrogen and ischemic/reperfusion (I/R) injury, Booth et al. (2005) designed an experimental study where they studied the cardioprotective role of ER-ɑ and ER-β on female rabbit hearts . After pThe mitochondria play a vital role in the overall integrity of a cell. Mitochondrial connexin43 (Cx43) is crucial to cardiac myocyte recovery following I/R injury . Wang etTo elaborate on the association of estrogen and congestive heart failure (CHF), Satoh et al. (2007) studied the estrogen-induced protection against CHF . After eZhang et al. (2018) studied the therapeutic properties of EXD (Er-Xian Decoction), commonly found in Chinese medicine to treat menopausal syndrome . After 1The counterplay of estrogen and progesterone in relation to myocardial I/R insult was studied by Booth et al. (2007) . InfarctOther studies done by Xu et al. (2004) and Luo et al. 2016) found estrogen-treated models to exhibit decreased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and decreased production of reactive oxygen species (ROS) respectively ,19, whic6 found eEstrogen receptor-ɑ (E2-ɑ or ER-ɑ)In an experimental study to understand the cardioprotective effect of E2-ɑ receptors, Xue B et al. 2007) studied the role of E2 in angiotensin II (ANG-II) induced hypertension [07 studieWang et al. (2006) studied the role of ER-ɑ in MI . MI causLeft ventricular outflow tract (LVOT) obstruction occurs in 15%-25% of TC patients leading to heart failure . WestphaEstrogen receptor-β (ER-β or E2-β)Multiple studies have been done to understand the cardioprotective role of ER-β. In one of the studies, Zhu et al. (2002) studied the effect of E2-β on hypertension . In theiTo further elucidate the role of E2-β on lowering blood pressure, Jazbutyte et al. (2008) treated the OVX mice with E2-β agonist (8β-VE2) . After 1Moreover, I/R injury leads to mitochondrial instability by the activation of pro-apoptotic cytochrome c, which in turn causes the activation of caspase 9 . SchuberAnother study done by Skavdahl et al. (2005) studied the role of E2-β in cardiac hypertrophy . Upon inCardiac hypertrophy is a precursor to diastolic dysfunction, and E2 has been associated with preventing cardiac hypertrophy . Pedram GPERTraditionally, it was thought that estrogen exerts its cellular response only through nuclear receptors, E-ɑ and E-β. However, recently a third kind of ER, GPER, has been found located in the endoplasmic reticulum that binds specifically to estrogen to produce nongenomic effects of estrogen . FredettTable TC is a reversible cardiomyopathy that impacts men and postmenopausal women more than premenopausal women. Estrogen seems to have a cardioprotective role in cardiomyopathies with a complex mechanism of action. The purpose of this article was to have a thorough understanding of this novel pathology. The studies mentioned in this article present us with strong pieces of evidence towards the cardioprotective role of estrogen with a positive impact on infarct size, cardiac hypertrophy, hypertension, heart failure, etc. A major limitation of this article is that all the studies included in this review are animal based, thus, there is a need to develop human studies to extrapolate these findings. Although estrogen replacement therapy has been known to be beneficial against vaginal atrophy and osteoporosis, a significant side effect of estrogen supplement treatment is the development of a hypercoagulable state resulting in deep vein thrombosis and potentially pulmonary embolism. Therefore, we recommend future studies focus on an estrogen regimen that would be beneficial to both sexes. The most prevalent theory about the mechanism of TC is that elevated levels of catecholamines lead to a hyperadrenergic state. However, our research could not find an article linking estrogen and the hyperadrenergic state of catecholamines. In addition, we strongly recommend including estrogen levels as part of the initial blood work for patients presenting with signs and symptoms of cardiac pathology.


The search for the ways of thermal stabilization of supported metal catalysts is an important challenge in the modern catalysis. Chemical modification of support seems to be the most versatile approach to stabilize the metal particles against sintering and alter their catalytic performance. Also for such modification nitrogen doping can be used and is considered rather perspective. In a recent manuscript  we have developed the procedure of N-doping of alumina thin film grown at the surface of metal substrate. Proposed N-doped model alumina support is suitable for catalysis – oriented surface science studies and improves the resistance of supported metal particles against thermal driven sintering. Herein, we provide useful complementary data for the characterization of the prepared materials in the form of:in situ / ex situ XPS (X-ray photoelectron spectroscopy) spectra at every stage of sample preparation, including angle resolved XPS experiments and thermal stability tests;STM (scanning tunneling microscopy) images of supported gold catalysts. Presented data support the proposed mechanism of film formation and modification. Specifications TableValue of the Data•These data are important for the substantiation of the mechanisms proposed for the formation and N – doping of alumina film on the surface of metal substrate.•Researchers in surface and materials science and catalysis can find the spectroscopy data, useful for characterizing analogous materials and application of such materials for catalyst designing.•Reported data can be used for the design of new catalysts based on N-doped alumina supports.1The data in this article refer to the samples described in “N-doping of alumina thin film support to improve the thermal stability of catalysts: preparation and investigation” 5_FCA-N_00_FCA_1 , 5_FCA-N_1)). In 6_FCA-N_1, 7 × 10−7 mbar NO exposition at 670°C) are shown. The plots in 5_FCA-N_1) confirms the reproducibility of angular dependences for N – doped films prepared by the proposed procedure. 3_FCA-N_0) and (3_FCA-N_1) . In 6_FCA-N_1), XPS spectra of N1s line before and after NO treatment, when NO presents in residual vacuum, are shown in Au_FCA-N), the survey XPS spectrum shows presence of Au4f line after vacuum vapor deposition of the gold on N-doped AlOx/FeCrAl. Au_FCA) and N-doped AlOx/FeCrAl (Au_FCA-N) samples confirms absence of restrictions for thermal sintering of particles  and short distance between particles).The survey XPS spectra show presence of main components of foil used as substrate for sample preparation  after foil cleaning procedure and before film formation (In XPS dataFiles in the data repository are organized in folders according to methods of sample's study: XPS (X-ray photoelectron spectroscopy) and STM (scanning tunneling microscopy). ‘XPS’ folder includes XPS spectrum data files (Excel files). Files are named according to the following convention ‘Fig X spectrum type sample name.xls’, where “X” is the number of the figure ; “spectrum type” is ‘survey spectrum’ or ‘region spectrum’ (regions are specified commonly for XPS); “sample name” is according to the 2The preparation of all samples is described in principle in “N–doping of alumina thin film support to improve the thermal stability of catalysts: preparation and investigation” 2.1A foil of aluminum-containing heat-resistant steel Fecralloy® alloy  in “as rolled” condition was used for the film preparation. The FeCrAlloy foil of this trademark was chosen as commercially available and inexpensive material. Use of such material as substrate allows reproducible preparation of unrestricted number of samples of supports and catalysts. According to the datasheet, the alloy composition is (wt. %): Fe – 72.8%, Cr – 22%, Al – 5%, Y – 0.1%, Zr – 0.1%. The thickness of the foil (1 mm) was chosen from the offered list to prevent bending of the samples and as result a damage of alumina film continuity during the further manipulations with sample (XPS to STM sample holder transfer and so on).“as rolled” rather than “annealed” foil condition since in this case the amount of dissolved oxygen is enough for formation of the initial alumina film over the surface of steel foil It is important to use 2.2Before the mounting on sample holder loading the samples were cleaned in ultrasound bath (96% ethanol) to remove technical contamination of steel rolling and shape machining.Prior to the film preparation both sides of the specimens were cleaned by argon sputtering to remove surface contaminations as well as the major part of the surface native oxides. The regimes of argon sputtering  were chosen to achieve Fe/C atomic ratio of ∼5 as a criterion for the quality of cleaning. It should be mentioned that the comparable fractions of metal and oxide aluminum have to be well seen after argon sputtering see . The pur2.3−7 mbar – 1 × 10−6 mbar.After cleaning procedure steel foil samples were used for preparation of N–doped alumina thin film support following procedure described in “N–doping of alumina thin film support to improve the thermal stability of catalysts: preparation and investigation” STM dataTo use resistive heating to anneal the sample tungsten wire legs (∅ 0.2 mm) on tantalum springs were spot-welded to the backside of the foil disks. Tungsten legs on tantalum springs prevent the distortion of the sample position during the annealing, which is important for precise angle resolved XPS experiments . The sam+ sputtering followed by vacuum annealing at 700°C for 20 min and final Ar+ sputtering was used as pretreatment to remove possible contaminations with low melting point. After that all the subsequent procedures were carried out at temperature lower than 700°C (except for the temperature stability tests) and 670°C was pointed as the highest recommended temperature of using the samples For all foil samples, a cycle of Ar2 were tuned up in a by-pass line by leak valves and then switched directly into an XPS analytic chamber to carry out in situ XPS experiments. This allowed controlling NO / O2 exposition precisely, which was important for the final film preparation, minimizing the time of NO / O2 flow stabilization when leak valve is used. The initial residual gas pressure in the chamber was ∼10−9 mbar, rising to 10−8 mbar between in situ experiments. The influence of NO presence in residual vacuum is shown in The flows of NO and O2.47/2 (binding energy BE = 84.0 eV), Ag3d5/2 (368.3 eV) and Cu2p3/2 (932.7 eV) peaks X-ray photoelectron spectroscopy study was performed at a VG ESCALAB HP electron spectrometer equipped with retardation grid in front of entry slit of hemispherical analyzer and single channeltron detector Ag3d5/2 38.3 eV an2.5STM experiments were performed on an UHV (ultrahigh-vacuum) variable temperature STM VT-7000 . The cut Pt/Ir tips were used for STM measurements. The quality of the tips was tested periodically by achieving the atomic resolution on highly oriented pyrolytic graphite. Both topography and tunneling current images were acquired simultaneously to recognize the regions of the different electron states (film and particles) or to check surface non-uniformity. The STM images were analyzed with WSxM 5.0 Develop 3.3 software Aleksey M. Dmitrachkov: Investigation, Data curation. Ren I. Kvon: Investigation, Writing – review & editing. Anna V. Nartova: Conceptualization, Methodology, Investigation, Data curation, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Three filter-based factor selection methods are executed, and leave-one-out cross-validation by linear discriminant analysis is measured on predicted scores of all models. Finally, the Monte Carlo simulation method with 10 iterations of repeated sampling for optimization of validation performance is applied to select the optimum model. The standard and proposed models are executed over simulated and real data sets for efficiency comparison. The PLSρs is found to be the most appropriate proposed method to model the observed ranked data set of antenatal care visits based on validation performance. The optimal model selected 29 influential factors of inadequate use of antenatal care. The important factors of reduced antenatal care visits included women's educational status, wealth index, total children ever born, husband's education level, domestic violence, and history of cesarean section. The findings recommended that partial least square regression algorithms coupled with rank correlation coefficients provide more efficient estimates of ranked data in the presence of multicollinearity.The frequency and timing of antenatal care visits are observed to be the significant factors of infant and maternal morbidity and mortality. The present research is conducted to determine the risk factors of reduced antenatal care visits using an optimized partial least square regression model. A data set collected during 2017-2018 by Pakistan Demographic and Health Surveys is used for modeling purposes. The partial least square regression model coupled with rank correlation measures are introduced for improved performance to address ranked response. The proposed models included PLS Kendall's τB rank correlation coefficient, the Stuart-Kendall τC rank correlation coefficient, Somers' delta (D), Goodman-Kruskal's tau τGK, Goodman-Kruskal's gamma (G), and Thiel's U correlation coefficient are proposed in this study. To the best of our knowledge, no previous research has considered multicollinear covariates in modeling the ranking of ANC visits of Pakistani women. Thus, the objectives of this study are twofold: (i) to develop a regression model for the ranked response covering the issue of multicollinearity and (ii) to determine the risk factors for inadequate use of antenatal care. This study introduced eight novel PLS algorithms addressing the concern of multicollinearity for a ranked response which is never discussed earlier. The proposed and standard algorithms are executed on a real-life application of ANC data for comparison purposes. These algorithms will facilitate users to obtain more efficient models than the standard PLS approaches for specifically ranked data. Regarding the clinical importance of this study, the influential selected variables of ANC will help maximize the chances for a normal pregnancy by providing priority interventions, increasing coverage, and improving health quality. The novel contribution of this study included:eight new PLS algorithms based on rank correlation loading weights are proposed to address rank responsethe significant factors of ANC utilization of Pakistani women are identifiedPakistan sets targets to minimize the maternal mortality ratio (MMR) to 140 per 100,000 live births by 2015 by increasing skilled birth attendants and improving access to reproductive health care as the fifth Millennium Development Goal (MDG) for improving maternal health suggested. The MDG progress assessment reported that Pakistan was not close to attaining the target in 2015. Recently, Pakistan has endorsed the Sustainable Development Goals (SDGs), committing to decrease the MMR to 70 per 100,000 live births by 2030 by increasing skilled birth attendance, facilitation to modern contraception, and extending coverage of health workers. The Government of Pakistan took initiatives and made good progress in maternal health indicators during the last decade, and a significant decline was reported in MMR from 276 to 178 –13. The y) which is ranked as inadequate (0-3 visits), intermediate (4-7 visits), and adequate (8-9 visits).The data acquired from Pakistan demographic and health survey (PDHS) 2017-2018 for Baluchistan is used for the present study. A total of 943 observations (women) with 43 factors are included in the analysis. The frequency of ANC is considered the ordinal response factor ( and suppose some C (where C ≤ p) to represent the number of components for prediction. Then, for c = 1, ⋯, C, the general algorithm executes aswc = Z′c−1yc−1The loading weights are tc = Zc−1wcThe score vector is Z-loadings (Pc) and Y-loadings (qc) by pc = Z′c−1tc/t′ctc and qc = y′c−1tc/t′ctc, respectivelyEvaluate Zc−1 and yc−1 by Zc = Zc−1 − tcp′c and yc = yc−1 − tcqcDeflate c < CRepeat the algorithm, if Consider the regression model W, S, P, and q are the matrices/vectors to compile the loading weights, scores, Z-loadings, and Y-loadings computed at each iteration of the algorithm, respectively. The regression estimators of the PLSR model are computed by Consider that =y¯−Z¯B^ . The geny but if the response is measured on a rank scale then this standard method may not work well. The most important phase of the PLS algorithm is to compute loading weight having the ability to choose significant factors. Loading weights compute the correlation between the dependent variable and predictors. If the data set is ranked then Spearman's rank correlation coefficient, Kendall's τA rank correlation coefficient, Kendall's τB rank correlation coefficient, the Stuart-Kendall τC rank correlation coefficient, Somers' delta (d), Goodman-Kruskal's tau τGK, Goodman-Kruskal's Gamma (G), and Thiel's U correlation coefficient are the recommended measures of rank correlation. These measures of association are used to compute the loading weights of the PLS algorithm. The modified loading weights of PLSR are visually displayed in The standard PLS is designed for continuous dependent variable ρ (ρs) [di denotes the difference between the two ranks of each observation and n is the number of observations and the modified PLSR algorithm is referred to as PLSρs.Spearman's rank correlation coefficient or Spearman's ρ (ρs)  is a nonτ coefficient is a measure of rank correlation. The tau-A (τA) will not make any adjustment for ties [nc is the number of concordant pairs, nd is the number of discordant pairs, and n0 = n(n − 1)/2, and the modified algorithm is named as PLSτA.The Kendall rank correlation coefficient or Kendall's for ties . It is uB makes adjustments for ties [τB asKendall's tau-for ties . The PLSn1 = ∑iti(ti − 1), n2 = ∑juj(uj − 1), ti is the number of tied value in the ith group of ties and tj is the number of tied value in the jth group of ties and the proposed model is termed as PLSτB.where τC replaced the weights of PLS as follows:The Stuart-Kendall (tau-c) is more suitable for contingency tables . The τC m is the minimun number among rows and coulmns, and the modified PLSR algorithm is called PLSτC.where D) [Y with respect to variable Z are defined asThe PLS loading weights based on Somers' delta(D)  of variaτ is symmetric, whereas Somers' D is asymmetric in Z and Y, and the model is named as PLS D.Kendall's tau τGK [Goodman-Kruskal's tau τGK  is integτGK.and the modified algorithm is called PLSG) [The estimate of Goodman-Kruskal's gamma (G)  is used nc is the number of concordant pairs and nr is the number of reversed pairs. Goodman-Kruskal's gamma drop ties, and the PLSR model is named as PLS G.where U correlation coefficient or uncertainty coefficient [Thiel's fficient  altered H(Z) represents the entropy of a single distribution and H(Z | Y) represents the conditional entropy, and the modified PLSR algorithm is referred to as PLS U.where Several variable selection methods integrated with PLSR have been introduced. The following are considered here.wj are used to measure of importance of predictors and are defined as [The loading weights fined as . (9)LW=wThe regression coefficient estimates are computed as (10)RC=WThe significance multivariate correlation measure is used to reduce the effect of irrelevant predictors and enhance the influence of significant variables included in the model. The SMC  is compuInitially, the PLS models with modified loading weights are executed for simulated data set for ranked variables. A sample of size 1000 with 100 predictors is generated. The response variable and 50% predictors are generated over 3 ranks, and the remaining explanatory variables are distributed over 4 ranks.A, Kendall's coefficient-B, Stuart-Kendall's, Somers' delta, Goodman-Kruskal's tau, Goodman-Kruskal's gamma, and Thiel's U coefficient are used as loading weights of the PLS algorithm to fit oversimulated data set to observe the variation in performance of standard and proposed models based on Akaike information criterion (AIC).Spearman's coefficient, Kendall's coefficient-τGK as modified loading weight showed optimum performance compared to eight other models without integrating any variable selection method. All other proposed models also evidenced higher efficiency compared to standard PLSR. Figures τGK and PLS U have optimum performance compared to all other models. The standard and modified models are also executed over the real data set of ANC for comparison of accuracy. The data set of ANC visits had 43 predictors sampled over 943 samples (mothers). The Spearman rank correlation coefficient is used to examine the multicollinearity in the data. The correlogram map measured strong correlation among 16 covariates while intermediate correlation among several other predictors is observed and shown in A, Kendall's coefficient-B, Stuart-Kendall's, Somers' delta, Goodman-Kruskal's tau, Goodman-Kruskal's gamma, and Thiel's U coefficient are used to modify loading weights. Then, three filter-based factor selection methods are performed on each of the nine PLSR models. Leave-one-out cross-validation by linear discriminant analysis is measured on predicted scores of all 27 models. Finally, the Monte Carlo simulation method is used with 10 iterations of repeated sampling for optimization of validation performance. The standard PLSR is compared with the PLSR integrated with Spearman's coefficient (PLSρs), PLSR integrated with Kendall's coefficient-A (PLSτA), PLSR integrated with Kendall's coefficient-B (PLSτB), PLSR integrated with the Stuart-Kendall (PLSτC), PLSR integrated with Somers' delta (PLS D), PLSR integrated with Goodman-Kruskal's tau (PLSτGK), PLSR integrated with Goodman-Kruskal's gamma (PLS G), and PLSR integrated with Thiel's U coefficient (PLS U).The frequency of ANC is classified into three ranks as inadequate, intermediate, and adequate. The ratio of 70 : 30 is used to randomly split data into training and testing sets, respectively. Initially, PLSR integrated with rank correlation coefficients as loading weights is executed. The Spearman's coefficient, Kendall'scoefficient-ρs and PLS D have optimum performance compared to standard PLSR and other proposed PLSR models for the observed data of ANC visits. The PLS τA and PLSτGK also have relatively higher accuracy than standard PLSR. In τA and PLSτB are more efficient in terms of optimization accuracy compared to standard and modified PLS models. A similar pattern of performance for PLSρs and PLSτC is observed compared to standard PLSR. Four other proposed PLSR models integrated with rank correlation coefficients showed slightly lower accuracy compared to standard PLSR.ρs, PLSτA, PLSτB, PLSτC, PLS D, and PLS U featured incremental performance after accounting RC selection method. Two other proposed methods PLSτGK and PLS G demonstrated approximately identical efficiency as standard PLSR. Standard and modified PLSR models embedded with the SMC factor selection method are compared in ρs and PLSτC evidenced higher accuracy than standard PLSR. The other four modified models including PLSτA, PLS D, PLS G, and PLS U measured analogous accuracy as PLSR.ρs is found to be the most appropriate proposed method to model the observed ranked data set of ANC. ρs compared to all other models without considering any variable selection method. Integrated with RC factor selection methods, the proposed PLSρs showed higher efficiency compared to standard PLS visualized in ρs established the highest optimization accuracy of nearly 78% among all other methods in 10 combined with the SMC factor selection method. Based on this evidence, PLSρs featured with the SMC method is finally picked for the selection of influential factors of ANC. For extraction of influential factors of ANC, PLSρs coupled with SMC is executed and estimates of 29 variables are presented in Comparison based on validation accuracy supported that PLSρs), PLSR integrated with Kendall's coefficient-A (PLSτA), PLSR integrated with Kendall's coefficient-B (PLSτB), PLSR integrated with the Stuart-Kendall (PLSτC), PLSR integrated with Somers' delta (PLS D), PLSR integrated with Goodman-Kruskal's tau (PLSτGK), PLSR integrated with Goodman-Kruskal's Gamma (PLS G), and PLSR integrated with Thiel's U coefficient (PLS U).To examine the significant predictors associated with ANC, sample data obtained from PDHS (2017-2018) is used. The occurrence of multicollinearity pointed to the application of PLS being a popular substitute for the standard regression model. Data is randomly divided into testing and training sets. Eight PLS algorithms established on rank correlation coefficients are introduced to address particularly the ranked response and compared with the standard PLS model to prove the improved efficiency in model building. The proposed models include Spearman's coefficient (PLSFurthermore, three variable selection methods are integrated with standard and modified PLS algorithms to estimate the accuracy to examine the variation in the performance of modified and standard PLS models with and without variable selection methods. The variable selection methods, namely, loading weights, regression coefficients, and significance multivariate correlation are considered here. The validation performance is computed for 10 iterations to examine the efficiency of nine PLS models integrated with variable selection methods.ρs is found to be the most appropriate proposed method to model the observed ranked data set of ANC. ρs compared to all other models without considering any variable selection method. Integrated with RC factor selection methods, the proposed PLSρs showed higher efficiency compared to standard PLS visualized in ρs established the highest optimization accuracy of nearly 78% among all other methods in 10 combined with the SMC factor selection method. Based on this evidence, PLSρs featured with the SMC method is finally picked for the selection of influential factors of ANC.Comparison based on validation performance supported that PLSρs and PLS D have optimum performance compared to standard PLSR and other proposed models without considering any factor selection method. For the observed data set, the PLSτA and PLSτB combined with the LW variable selection method are found to be more efficient in terms of optimization accuracy compared to standard and modified PLS models. Integrated with RC method for variable selection, the PLSρs, PLSτA, PLSτB, PLSτC, PLS D, and PLS U featured incremental performance compared to standard PLS. The PLSτGK and PLS G are found to exhibit approximately similar efficiency as standard PLSR. The PLSρs and PLSτC embedded with the SMC factor selection method evidenced optimum accuracy compared to standard PLS. Considering all validation comparisons, it is noticed that the modified models integrated with rank correlation coefficient exhibit higher efficiency for ranked data set of ANC compared to the standard PLS algorithm. The PLSρs coupled with SMC is suggested for modeling the ANC ranked data and 29 influential factors are observed to discriminate the ANC ranks. The proposed algorithms for rank response will facilitate researchers to address the regression models more efficiently even in the presence of multicollinearity in different fields of research. Since the rank response is specifically addressed rarely, the findings of this study offer new, potentially useful information for this ranked population. In the future, these algorithms may be integrated with other variable selection methods to observe the efficiency. Also, the proposed study can be extended for neutrosophic statistics [Regarding validation accuracy, very important and interesting facts are observed about the comparison of efficiency for ranked data. Primarily, PLSρs coupled with SMC identified the significant predictors of ANC using the optimized model for the observed data. The modified PLS models have the ability to address multicollinear ranked data more effectively. Regarding the clinical importance of this study, the influential selected variables of ANC will help maximize the chances for a normal pregnancy by providing priority interventions, increasing coverage, and improving health quality.Proposed PLS algorithms integrated with rank correlation coefficients are observed to be a better option with regard to model efficiency and variable selection of ranked simulated and real data sets. This suggests that these rank measure-based PLS algorithms provide models with superior potential. The PLS


To perform a reliable analysisof the SERIA spectra of these peptides, curve fitting of these spectrain the spectral region above 1500 cm This leads toa limitation of the application range of vibrational spectroscopybased on the detection of chemical traces . To overcome these limitations,surface-enhanced techniques of this method have been developed andapplied using highly concentrated fields in the vicinity of resonantlyexcited plasmonic structures. The surface-enhanced technique alsoovercomes another limitation of infrared spectroscopy, where the extremelyhigh IR absorption of water prevents the direct use of an aqueousmedium in IR measurements since the enrichment of the sample alongthe metal surface reduces the water content in the observed volume.Vibrationalspectroscopy (infrared absorption and Raman) is a widelyused, reliable, and powerful method for studying conformational changesand molecular interactions and for unambiguously identifying and characterizinga variety of molecules by their vibrational fingerprint. However,in conventional form, it does not provide sufficient sensitivity fortrace concentrations and thin molecular layers  developed in 1974.2 To date, however, SEIRA has not gained the importanceof SERS due to the lower signal enhancement (compared to SERS),3 which is typically 101–103 when the molecule is adsorbed on or near (10 Å or less)rough surfaces of a variety of metals. The first SEIRA studies usedmainly noble metals . Later reports showed the possibility of using other metals,14 semiconductors,15 and polar dielectricnanostructures.16In the early 1980s, Hartstein, Kirtley, and Tsang first observedthe phenomenon of surface-enhanced infrared absorption,5 periodic array-based substrates (consisting ofparticles and holes),17 and less frequentlymetal sols.18 Metal films consist of growingand converging isolated particles that eventually form a continuousfilm. During this process, the signal from the adsorbate is stronglyenhanced until the percolation threshold is reached (or close to it),whereupon the signal strength decreases until it completely disappearsonce a continuous film is formed.19 Signalenhancement on these substrates is mainly based on the off-resonancemechanism. For resonance enhancement to take place in the infraredregion, the structure must have the right size, which unfortunatelyis not possible for metallic island layers . The solution in this situation is to use other metalsubstrates, such as metal sols, which are also important for otherreasons; e.g., they are relatively fast, easy, and inexpensive toobtain; they allow reproducibility of signal enhancement due to synthesisprocedures that ensure low dispersion of the nanoparticle diameterin the sol and do not require strict topological control; they canbe used in transmission mode without using complicated optical systems,and the sample is attached to a surface of colloidal nanoparticlesbefore measurement. The latter is particularly important in the caseof metals with photocatalytic properties, whose properties can beinhibited by functionalizing their surface with biological material,and in the context of the development of hybrid biodevices (biomoleculesassociated with the substrate that actuates them). This concept assumesthe possibility of triggering and testing the properties of the adsorbateat the interface created between the biomaterial and the substrate.However, despite numerous studies on optical biosensing with SEIRA,there are still too many unknowns  that preclude routine use of this technique inbiology and medicine. For this reason, we have undertaken the currentresearch to achieve improved absorption using commonly available andhomogeneous, in terms of shape and diameter, Ag and Au colloids (dueto the aforementioned advantages), which will allow the broader androutine application of SEIRA. At the same time, we extend relativelylittle knowledge about the use of SEIRA for the study of biologicalsystems, the detailed properties of which we present. The choice ofbiological systems such as neuromedin B, bombesin, neurotensin, andbradykinin was dictated by the fact that they are the natural ligandsof metabotropic seven-transmembrane G-protein-coupled receptors (GPCRs),which are overexpressed on the surface of many malignancies, makingthese receptors  potentially available as receptor-positive cancermarkers in early diagnosis for tissue lesions detection and anticancertherapy.21The SEIRA effectis mainly studied on chemically deposited andvapor-deposited metal island films, nanoparticle decorated films,22 which usually contain carbonyl and thiol groups23 or adsorbates in the form of small and/or symmetricmolecules28 and less frequently thin polymer films.34 Few literature reports indicate the use of SEIRA in the study ofbiosensors,51 in cancer drug research (cis-platinum and doxorubicin),52 and as a diagnostic criterion for cancer.53The supplemented information in thedatabases on spectroscopy ofamino acids and neurotransmitters will also allow a more accurateinterpretation of the spectra of complex molecules, such as peptidesand proteins. This is because many research groups have focused onthe preparation of new substrates and the determination of signalenhancement and SEIRA mechanism using adsorbates,54 The enhancement of these bands depends on threemain factors, which have been classified as “chemical”and “physical” based on their origin.17 Physical factors are associated with an enhancement ofthe electromagnetic field near a rough metal surface or metal islandfilms and can be divided according to whether the frequency of theplasmon matches the frequencies of the adsorbate vibrations (on-resonancecontribution) or whether the plasmon frequency is far away and onlya small direct interaction between plasmons and vibrations occurs.17 In the infrared region, the frequency of theplasmon can be tuned by morphology  of the metal islands/layers, the surface structureof the supporting substrate, the experimental conditions, and thesurrounding medium.56 In the case of chemical factors, it is assumed that chemical bondingleads to changes in the electronic structure of the adsorbate andthus changes the dipole transition moment of the adsorbate vibrations,making vibrations not allowed in the infrared spectrum infrared active.SEIRA is concerned with those bands of the adsorbate whosevibrationalmodes contain a dipole component perpendicular to the surface.57Other factors affecting infrared absorption include the chemicalcomposition of the adsorbate; infrared absorption occurs mainly forvibrations of polar groups with large dipole moment gradients. Becauseof this phenomenon, this work also extends the knowledge of previouslypublished works on SERS sensing.Unprotected amino acids werepurchased from Sigma-Aldrich, Poland, and used without further purification.Supporting Information for details).Neuromedin B (NMB), bombesin (BN), neurotensin(NT), and bradykinin (BK) were synthesized via the solid-phase methodusing the Fmoc strategy and starting from Fmoc-Wang resin   and 40 nm (Ag)) were purchased from Merck(Poland).Before SEIRA measurements,each peptide (30 μL of an aqueous peptide solution) was immobilizedon colloidal suspension (10 μL). The mixture peptide/colloidalnanoparticles were then deposited on a diamond ATR adapter and allowedto dry. Unbound peptide molecules were removed through washing withdeionized water and allowed to dry. The process was repeated threetimes.–1 and 128 scans. SEIRA spectra were recorded three times at threedifferent locations on each substrate surface. The SEIRA spectra ofa given adsorbate on a given substrate were almost identical exceptfor small differences (up to 5%) in some band intensities. No spectralchanges that could be associated with the decomposition of the samplewere observed during these measurements.The spectra were recorded on an FTIR Thermo ScientificNicolet 6700 spectrometer equipped with a diamond ATR accessory. Measurementconditions were the following: a resolution of 4 cmMultiple bands not separated werefitted using a GRAMS/AI program .Briefly, a 50/50% Lorentzian/Gaussian band shape for all bands wasassumed and fixed. The number of bands, their initial wavenumbers,bandwidths , and intensities were selectedbased on results from previously published IR studies and carefulexamination of spectra obtained in this work.3D peptide structures were obtainedby GaussView 3.0  and UCSF Chimera1.8.1. software.l-cysteine (Cys), cystine, and l-methionine (Met)) immobilized on the surface of AgNPs and AuNPs.ATR-FTIR spectra are also included in this figure to highlight thechanges between SEIRA and ATR-FTIR spectra. The SEIRA spectra in thespectral region below 1000 cm–1 are not analyzedin detail; instead, the observed bands and their assignments are summarizedin 63 However, there are some differences in the intensity and positionof the bands. The ATR-FTIR spectra have much stronger bands at lowerwavelengths than at higher wavelengths compared to the transmissionFTIR spectra. This is because the penetration depth  depends on the radiation wavelength and increaseswith increasing wavelength. The wavenumber shift results from theamount of reflected radiation, which depends on the different refractiveindexes of the IRE crystal and the sample at different frequenciesof the interacting light. Shifts in band positions are thus opticaleffects caused by changes in the refractive index.64–1. This method is advantageous for highlighting small relative shiftsin the wavenumbers of bands and allows the separation of overlappingbands.–1).Since dipole moments of polarbonds such as O−H, C=O,and N−H  changethe most during the vibrations of the molecule, they produce strongbands in the infrared spectra, most of which can be observed in theSEIRA spectrum of Cys on AuNPs (–1 [ν(C–S)of PH-T conformer]  and 1511c cm–1 [(ρsymb(NH3+)]  (where c denotes curve-fitted bands). The curve fit also indicatesthat 1511c, 1482c [(ρsymb(NH3+)], 1380c [νsym(COO–)], 1360c [δ(CH)], and 1326c cm–1 [(ρw(CH2)] SEIRAsignals increase in intensity compared to the corresponding ATR-FTIRbands. In contrast, 1423 [ρb(CH2)], 1064[ρipb(SH)], 870 [ρw(COO–)], 661, 634 [ρw(COO–)], 533 [δoop(COO–)/ρb(CH–SH)],and 448 cm–1 [ρb(CH2–CH–SH)] spectral features decrease in intensity. Twomore bands at 987 [ν(CC)/ρs(NCH)] and 513 cm–1 [ρb(CH2–CH–N)]appear in the SEIRA spectrum of Cys on AuNPs. On the basis of theabove information, it can be concluded that Cys is adsorbed on thesurface of AuNPs: (1) mainly via the −NH3+ group, (2) the −COO– and −SH groupsare involved in the interaction of Cys with AuNPs, and (3) as a resultof adsorption, a conformational change of the thiol group occurs.The presence and intensity (higher than that in the ATR-FTIR spectrum)of the 2541 cm–1 band indicate that the thiol groupon AuNPs is not deprotonated, and the free electron pair on sulfurhas contact with the AuNPs surface, which means that the C–Sbond (knowing that sulfur has sp3 hybridization) is tiltedtoward the AuNPs surface.For Cys deposited on the AuNPs surface(c [ρsymb(NH3+)], 1511c, 1404c [ρs(CH2)], 1381c, 1336c [ρsymb(NH3+)], and1295c cm–1 [ρw(CH2)] lose their SEIRA intensity most significantly. The ATR-FTIRbands at 2549, 823 [ν(C–C/S)/ρs(CSH)],753 [ν(C–S)Pc-G],and 636 cm–1 [ν(C–S)PH-T] disappear. A new weak SEIRA band at 613 cm–1 [ν(CH–COO–)] appears. Some other bands shift in wavenumber roughlymaintaining their intensity, e.g., 1141ATR-FTIR →1125SEIRA cm–1 [ρr(NH3+)], 940ATR-FTIR → 962SEIRA cm–1 [ν(N–C)], 865ATR-FTIR → 845SEIRA cm–1, 692ATR-FTIR [ν(C–S)Pc-G] → 674SEIRA cm–1 [ν(C–S)PH-T]. Given this, it appears that the thiol group ofCys, which adopts one conformation (the same as on the AuNPs surface),is deprotonated on AgNPs and the contact between the amine/carboxylategroups and the surface of AgNPs is weakened.More spectral differences can be seenbetween the ATR-FTIR spectrum than theside chain of Cys. Therefore, bands with similar vibrations are expectedin the SEIRA spectra of cystine and Met. In general, the SEIRA spectraof cystine on AuNPs (c [(ρasymb(NH3+)] and 1570c cm–1 and in the spectralregion below 1350 cm–1 show higher and similar SEIRAintensities as the corresponding ATR-FTIR intensities except for theweak bands at 1245 [ρt(CH2)], 649 [ν(C–S)PH-T], and 573 cm–1 [ρt(NH3+)], which appear only in the SEIRA spectrumof cystine adsorbed on the surface of AuNPs. In the above spectralregion, for cystine on AgNPs, the SEIRA signals are weaker than thecorresponding bands for this amino acid deposited on AuNPs exceptfor the spectral feature at 1090 cm–1 [ρr(NH3+)], which has a comparable intensityin all cystine spectra shown. Bands at 1616c and 1577c cm–1 are the strongest for cystine on AgNPs,while bands at 1482c and 1453c cm–1 are the weakest for cystine on AgNPs. All these changes indicatethe following: (1) the presence of cystine in one rotamer on AgNPs and two C–Srotamers on AuNPs (at 757 and 649 cm–1) while maintainingthe same C–S–S–C fragment conformation  as in aqueous solution, as evidenced by the 538 cm–1 band of the trans–gauche–trans (TG′T) conformer, (2) a strong interaction betweencystine and the surface of AuNPs, and (3) a weak interaction of cystinewith AgNPs. A slightly larger shift in the wavenumbers for cystineon AuNPs  compared to cystine on AgNPs  confirms the differences in the strength of the interaction betweencystine and metal surfaces used.Two other sulfur-containingamino acids differ from Cys in thatcystine is an –1 band. The fwhm of this band decreases dramatically in the SEIRAspectra , and the band shape becomessymmetric compared to the asymmetric shape of the corresponding ATR-FTIRband. The second derivative of the Met ATR-FTIR spectrum shows threecomponents hidden under this band: i.e., one intense at 1511 cm–1 and two shoulders at 1497 and 1487 cm–1. According to work by Cao and Fisher, these bands can be assignedto the ρsymb(NH3+) modes ofdifferent conformers of the −H2C–S–unit.62 It can be concluded that the sidechain of Met in the solid-state adopts multiple conformations of the−H2C–S– unit, whereas it exists asonly one rotamer for the Met adsorbed on both metal surfaces.One of the distinguishing featuresof ATR-FTIR  moreintense than the other two bands from the envelope of the overlappingbands ], 1150, 1118 [ν(C–N)/ρs(CNH)], 980, 874, and 542 cm–1 are clearlyvisible. All these bands are due to the vibrations of the N-terminalgroup, and therefore this fragment is responsible for the direct interactionof Met with the surface of the two metallic NPs, which is slightlystronger for Met on AgNPs than for Met on AuNPs. However, the weak1632 and 539 cm–1 bands might indicate that thecarboxyl group is located at some distance from the metallic surfaces.Other significant spectral differences relate to the intensityof the bands. For example, the intensity of the overlapping 1606,1582, and 1560 cml-Histidine (His) SEIRAspectra ], 1620c [νasym(COO–)], 1583c [ν(ring) + ρipb(N1H)], 1520c [νasym], 1493c [ν(ring) + ρipb(N1H)], 1435c [δ(CH2) + δ(N1H)], 1402c [νsym(COO–)], 1341c [δ(N1H)], and 1321c cm–1 [ρt(N1H)] bandsare mainly enhanced. The wavenumbers of these SEIRA signals indicatethe zwitterionic form of His with a neutral imidazole ring (N1-protonated).66 Of these bands, the spectral features at 1591 and 1493 cm–1 are more intense on AgNPs compared to AuNPs, and the bands at 1357[ν(C−N)ring] and 668 cm–1 [ρoopb(ring)] appear only for His adsorbed on AgNPs.It can be concluded that the same molecular fragments of His interactwith the surface of both substrates, but either the strength of Ag/Au···N1–H interactions or the arrangement of the imidazolering to the substrate surface is slightly different on the two surfaces.In the SEIRA spectra of His on AuNPs  shown in 60 Spectral differences between this spectrum andthe SEIRA spectra of Phe adsorbed on AuNPs ], 1408c [νsym(COO–)], 1325c [ρoopb(CH2)], and 523 cm–1 (ρr(COO–) + δ(C=O)], respectively. Inthe spectrum of Phe on AuNPs, the 1547c [ρasymb(NH3+)] and 1524c cm–1 [ρsymb(NH3+)] SEIRA signalsare also observed, indicating a protonated amino group that helpsin the adsorption of Phe on the AuNPs surface.The ATR-FTIR spectrum of 68 the wavenumbers of the ν8a (at 1606c cm–1) and ν8b (at about 1570c cm–1) modes in theTyr spectra shown in –) form. The 1245 cm–1 spectral feature attributed to the ν(CPh–O) [ν7a] vibrations confirms theprevious statement, although TyrO– does not undergochemisorption on the surface of AgNPs and AuNPs, either via the π-electronsystem or via the free-electron pair at the phenolic oxygen. Thisis because no significant shift in wavenumbers (ATR-FTIR vs SEIRA)is observed (Δν = −2 cm–1) andthe AgNPs/AuNPs···TyrO– interaction is a sufficient reason for the broadening of the SEIRA bands(ΔFWFM = 7–9 cm–1). The amino and carboxylategroups of Tyr also interact with AuNPs ] and at approximately 1734c [ν(C=O)], 1415c [νsym(COO–)], 649 [ρω(COO–)], 573 [ρipb(NH3+) + δ(COO–)], and 529 cm–1, respectively. However, there are differences in the intensity ofthe bands due to the amine and carboxylate groups and phenyl ringvibrations for TyrO– on AgNPs compared to thosefor TyrO– on AuNPs. Briefly, 1243, 1329, 1362, and1415 cm–1 bands in the spectrum of TyrO– on AgNPs are 3 times less intense than the 1587 cm–1 SEIRA signal, and the bands in the wavenumber region below 900 cm–1 show even lower intensity, while the 1586 cm–1 band in the spectrum of TyrO– onAuNPs is less than 2 times more intense than the bands assigned tothe −COO– and phenyl ring vibrations. Thesefluctuations indicate that Tyr interacts directly with the AgNPs surfacevia the −NH3+ group. Moreover, M. Osawaet al. have shown that when the – COO– groupis bound to the metal surface as a bidentate coordination ligand,the νs(COO–) mode is enhanced,while the νas(COO–) mode is notenhanced compared to the same bands in the IR spectrum.69 From these results, it can be concluded thatthe bidentate coordination of the C-terminal groupof TyrO– is present on both metal surfaces.Considering thevarious possibilities of Tyr adsorption on metallicsurfaces,–1 in the ATR-FTIR spectrum of l-tryptophan (Trp) (c [ρsymb(NH3+)], 1449c , 1414c, 1356 ,and 743 cm–1 .70 In the wavenumber range of 1700–1500cm–1, the intensities of the 1665c [ρasymb(NH3+)] and 1643c cm–1 [νasym(COO–)]bands increase significantly in terms of the ATR-FTIR intensity, whilethe 1590c [W2], 1545c , and 1517c cm–1 [ν(pyrrole)] SEIRA signals decrease significantly in intensity.On the basis of the above observations, it can be assumed that Trpadsorbs on AgNPs via the terminal groups and pyrrole nitrogen of theindole ring. On the other hand, in the SEIRA spectrum of Trp on AuNPs(c and 1615c cm–1 [ν(phenyl)]bands increase in intensity, the 1558c, 1519c, 1351, and 740 cm–1 bands decrease in intensity,and 1449c and 1316 cm–1 [ν(pyrrole)]SEIRA signals disappear compared to the ATR-FTIR spectrum. Thus, Trpinteracts with the surface of AuNPs via the −COO– and −NH3+ groups and the phenyl ringof indole.The comparison of the spectral region below 1500 cm73 and the resultsof the SEIRA studies are not available. Structural information forthese peptides can be obtained by analyzing the amide bands, particularlyamide I, II (of relatively strong infrared intensity), and III (Raman-active),whose wavenumbers are sensitive to peptide chain conformation and hydrogen bonding in the peptide backbone.There are fewliterature reportson the IR studies of the tested peptides,–1) is greater than the distance betweenthe maxima of adjacent bands. As a consequence, the individual componentbands cannot be separated in the experimental spectra. The curve-fittingprocedure for these regions allowed us to increase the separationof the overlapping components present in the broadband envelope. Forthe SEIRA spectra of NMB on AuNPs ring (Phe/Trp) vibrations. Considering that in the spectrum ofNMB on AgNPs it is difficult to identify other bands attributed toimidazole vibrations, while the phenyl (co)ring vibrations give spectralfeature at 1607c, 1547c, and 1506c cm–1, the intensity and width of the band at 1581cm–1 suggest that it is associated with the phenylring vibrations rather than the imidazole ring vibrations.As shown in  peptide 2, these c cm–1 forNMB on AuNPs and at 692 and 745 cm–1 for NMB onAgNPs suggest that the C=O group of Asn and the −S–CH3 fragment of Met interact with AuNPs and AgNPs, respectively.Thus, using the determined NMB structure , and the assignment of the remainingeight bands is as follows: δ(NH2), amide I (randomstructure), amide I (α structure), δ(NH2),W1, amide II, and W3. This band assignment again suggests that Asnand Trp are in contact with AuNPs. The SEIRAsignals at 1739ture see 4, it canc, 1684c, 1661c, 1649c, 1627c, 1589, 1548c, 1527c, and 1515c cm–1 and the 1701c, 1678c, 1660c, 1647c, 1626c, 1546c, 1525c, and 1498c cm–1 SEIRA signals in the spectra of BN, depositedon AuNPs (–1) also suggest that Met is close to the surface of AgNPs and theMet and His side chains are on the opposite side of the peptide chain. On the basis of theabove observations and considering the proposed BN structure , whereas TyrOH is close to the AgNPssurface (at 1620c and 1599c cm–1).For NT onAuNPs  of the C-terminus.Assuming an interaction between the C=O group and the AuNPssurface, one can expect bands assigned to the peptide bonds (at 1651c and 1541c cm–1) and to the vibrationsof the guanidine moiety , which is the case. The lastof the calculated bands (at 1601c cm–1) indicates the presence of the phenyl ring in contact with AuNPs.Similarly, for BK on AgNPs (–1 indicate the involvement of the phenyl ring in the peptide interactionwith AgNPs. The absence of ν(C=O) indicates that BK adoptsan orientation on the surface of AgNPs in which only the guanidineand the phenyl ring interact with AgNPs  and the Trp ring are in contact with the surfacesof both metals, but the amidated C-terminus or sidechain of Met interacts with AuNPs and AgNPs, respectively. In thecase of BN, the indole and imidazole rings, the amidated C-terminus, and the peptide bond(s) are responsible for the interactionwith AgNPs, while the side chains of Trp and Met and the peptide bond(s)are on or near AuNPs. The side chains of Tyr and Arg interact withboth metallic surfaces but in a different state of protonation ofthe hydroxyl group (TryO– on AuNPs and TyrOH onAgNPs). Phe, Arg, and the C-terminus are responsible for the adsorptionof the last peptide studied (BK) on the surface of AuNPs, but Argand Phe are in contact with AgNPs.In this work,we present SEIRA results for the above neurotransmittersimmobilized on the surface of readily available and homogeneous, interms of shape and diameter, Ag and Au nanoparticles, which may bringus closer to the more frequent and routine application of SEIRA. Analysisof SEIRA spectra of peptides was possible because of curve fittingof these spectra and SEIRA data for selected amino acids . On this basis, it was shown that peptides adsorb differentlyon the two metal surfaces via molecular fragments located on the C-terminal part of the peptide chain; similar conclusions were drawnfrom the studies of the biological activity of these peptides.87 Thus, evidence has been obtained confirming the validity of SEIRAto select those peptide fragments within the study group of peptidesthat play a role in the substrate–receptor interaction in systemswhere biological studies are difficult or do not lead to the unambiguousdetermination of the peptide fragments responsible for its biologicalactivity.The SEIRA results also showedthat the investigated peptides changetheir structure upon adsorption and interact with the surfaces ofAuNPs and AgNPs with amino acids residues located in the