To evaluate the performance and to identify predictive factors of performance in prevention of mother-to-child HIV transmission programs (PMTCT) in sub-Saharan African countries.From 2000 to 2011, PMTCT programs included in the Viramune Donation Programme (VDP) were prospectively followed. Each institution included in the VDP provided data on program implementation, type of management institution, number of PMTCT sites, key programs outputs . Nevirapine Coverage Ratio (NCR), defined as the number of women who should have received nevirapine , was used to measure performance. Included programs were followed every six months through progress reports.A total of 64 programs in 25 sub-Saharan African countries were included. The mean program follow-up was 48.0 months (SD = 24.5); 20,084,490 women attended in antenatal clinics were included. The overall mean NCR was 0.52 (SD = 0.25), with an increase from 0.37 to 0.57 between the first and last progress reports (p<.0001); NCR increased by 3.26% per year-program. Between the first and the last report, the number of women counseled and tested increased from 64.3% to 86.0% (p<.0001), the number of women post-counseled from 87.5% to 91.3% (p = 0.08). After mixed linear regression analysis, type of responsible institution, number of women attended in ANC, and program initiation in 2005-2006 were significant predictive factors associated with the NCR. The effect of the time period increased from earlier to later periods.A longitudinal assessment of large PMTCT programs shows that scaling-up of programs was increased in sub-Saharan African countries. The PMTCT coverage increased throughout the study period, especially after 2006. Performance may be better for programs with a small or medium number of women attended in ANC. Identification of factors that predict PMTCT program performance may help in the development and expansion of additional large PMTCT services in sub-Saharan Africa. As home to more than two-thirds of the global population of individuals living with HIV/AIDS, sub-Saharan Africa has the highest HIV/AIDS burden of any region in the world. Of children newly infected with HIV in 2011, 90% lived in sub-Saharan Africa  of syrup to be taken within 72 hours after delivery) and the number of new PMTCT sites opened since the prior report.A PMTCT coverage indicator was calculated in order to assess the overall performance of each program. This indicator, the NVP coverage ratio (NCR), was defined as the observed number of women receiving sd-NVP divided by the expected number of women who should have received sd-NVP. This denominator was calculated as the number of women attending ANC visits multiplied by the observed HIV seroprevalence .The proportion of women attending an ANC who participated in specific components PMTCT services  was also calculated.Descriptive statistics are given as means with their standard deviation (SD) for continuous variables and percentages for categorical variables. For each program, the cumulative incidence of follow-up in program-months was calculated. In order to assess the performance of the included institutions, univariate analysis was performed to identify potential predictors of the final NCR and the intermediate process indicators . To do so, average numbers computed for the whole study period for each program were used. Anova or non parametric Kruskall-Wallis tests were used for comparisons of continuous data, depending on the normality of variable distribution; the following factors were considered: time period of the first progress report , type of responsible institution , mean number of PMTCT sites , and mean number of women attended an ANC per month . For the indicators of PMTCT services, the mean difference between first and last progress report was assessed. We used multivariate linear mixed models to assess the independent effect of each predictor of the NCR entered as the dependent variable, using all individual progress reports available instead of aggregating data. The institution was modeled as a random effect so as to account for the longitudinal structure of the data based on repeated measures. Candidate predictors for multivariate analysis were defined a priori, based on the hypothesized relationships to be tested. In addition to the predictors previously described, time since program initiation was entered as an independent variable and interactions were tested between this latter factor and the other predictors, in order to check for varying temporal evolutions according to the predictors. Beta coefficients are given with their 95% confidence intervals (CI), indicating the adjusted difference in NCR (0–100) between categories for qualitative variables or per 1-point difference in predictor value for continuous variables. A two-tailed p value <0.05 was considered to be significant. Statistical analyses were performed using Stata 11.0 software package .There was no external source of funding for this research. Axios International is a commercial company, which has been responsible for managing and monitoring the Viramune Donation Program and distributing nevirapine (Viramune). Boehringer Ingelheim contracted the services of Axios International to manage the VDP. Boehringer Ingelheim had no role in study design, data collection, data analysis, data interpretation, or manuscript development. The corresponding author had full access to all data in the study and had final responsibility for the decision to submit for publication. All data analyses were conducted under the authorization from Boehringer Ingelheim.From 2000 to 2011, a total of 64 programs in 25 sub-Saharan African countries were implemented under the VDP and described in Between the date of initial inclusion in the VDP and submission of the final progress report, for the 64 programs, the mean program follow-up was 48.0 months  . Mean foDuring the study period, the mean number of women attending an ANC per program-month was 5,493 . The mean number of women attending an ANC per program-month varied among programs managed by institutions: 7,659  for programs managed by governmental institutions, 3,194  for NGOs and 5,076  for IAs (p = 0.07).For each of the PMTCT services indicators assessed, activity increased between the first and the last progress report. The mean number of women attending an ANC, pre-counseled and tested, post-test counseled, and receiving sd-NVP increased by 9,125 women per program-month , 6,699 per program-month , 4,689 per program-month , and 353 per program-month (SD = 900), respectively. The mean number of infants receiving sd-NVP increased by 269 per program-month (SD = 686), and the mean number of new PMTCT sites opened increased by 11.4 sites per program-month (SD = 32.1).The results of multivariate analysis of predictive factors associated with NCR are shown in This large longitudinal study comprises a diverse set of 64 PMTCT programs in 25 sub-Saharan African countries with median follow up of 45.8 months. Over the course of the VDP, the NCR increased by 3.26% per year-program, the mean number of women attending an ANC and undergoing pre-test counseling, HIV testing, and post-test counseling increased significantly, as did the number of women and infants receiving sd-NVP.The sd-NVP coverage was higher for programs that were initiated at later dates, as shown by a significantly higher NCR for programs starting in 2005–2006 compared with earlier time points. This suggests that PMTCT programs initiated later may have learned from the experiences of programs already implemented. External factors may also have increased overall awareness of and demand for such programs and driven additional women to attend an ANC, including ANCs participating in the VDP. These factors include an overall increase in access to healthcare, availability of healthcare resources and tools, and lessons learned from a variety of PMTCT and other access programs including those implemented by the Elizabeth Glaser Pediatric AIDS Foundation, the President’s Emergency Plan for AIDS Relief (PEPFAR), and donations from various governmental and philanthropic organizations .The overall NCR over the course of this study was 52%, and NCR increased throughout the study period, especially after 2006. The maximum NCR of 66% in 2009 is quite similar to the 59% (CI = 53%-66%) coverage of PMTCT services estimated by UNAIDS . Our finThe NCR was higher for programs managed by NGOs and IAs compared with those managed by governmental institutions. The type of managing organization appears to be a significant predictive factor for increased NCR. Several factors may account for this observation. First, the mean number of women attending an ANC was highest for sites managed by governmental organizations compared with NGOs or IAs, while programs with more than 100 women attending an ANC per month had significantly lower NCR than programs seeing fewer women each month. This suggests that the governmental sites may have had larger infrastructure or logistics challenges due to their need to meet greater demand than sites managed by other types of organizations. Consequently, the reduced NCR associated with government-run programs may be associated with the availability of resources  rather than technical competency. Second, NGOs and IAs are usually smaller than government organizations, with fewer key decision makers and layers of regulations, which may allow them to move more quickly in developing and mobilizing resources, as well as implementing and modifying programs. Thus, increased NCR for these organizations may be independent of the ability of specific types of institutions to execute PMTCT programs effectively. Moreover, because NGOs and IAs were permitted to participate in VDP with the approval of the government(s) in which their programs were to be conducted, it is likely that these organizations had governmental support that served as an additional asset in their efforts to deploy programs effectively.The rapid increase in the number of new PMTCT sites added per month between the first and last progress report suggests that programs had the operational capacity to develop and expand access to their PMTCT services. Strengthening operational capacity will be important for building PMTCT strategies that are integrated with routine care of women and children and HIV care and treatment programs, and for providing a suite of services and innovative approaches that can reduce MTCT –32. HoweThere is great potential for PMTCT programs to have a significant impact on reducing the burden of HIV/AIDS and improving health outcomes, especially for children, in sub-Saharan Africa , 24, 33.Our study has several limitations that should be noted. Longitudinal data collection and monitoring were difficult over the life of the programs. However, the VDP program manager regularly assessed the reporting data system, and reporting requirements were communicated to the program manager at the time the program was included in the VDP. Data were reported as expected and consistent with requirements, adding to the strength of the data set. Another limitation is that we could not examine potential factors that might explain observed patterns, such as variables related to socioeconomic resources, rural/urban setting, type of health facility , access to health care, and quality of care. Funding from international programs and initiatives also could have been potential factors leading to better performance over time. A third limitation derives from the fact that we cannot confirm that our results are representative of all PMTCT programs implemented in sub-Saharan Africa, particularly those based on different ARV regimens. We were also not able to determine if a pregnant woman actually took the sd-NVP dose she was given in PMTCT services, or if she administered the dose to her newborn infant because we lacked the ability to directly measure the drug compounds contained in cord blood samples. Identification of the date of program initiation as a significant predictor of NCR is also a limitation given that a past date cannot be used to predict performance of programs initiated since that time. However, as noted above, several factors associated with the 2005–2006 period  could be relevant for the design and implementation of future PMTCT programs. Finally, our findings rely on data covering an early era in large-scale PMTCT programs, when simpler antiretroviral regimens were used as opposed to the multi-drug regimens currently recommended. We believe that it is important to document the barriers encountered in interventions as simple as those based on single-dose NVP, because it is likely that such factors may also apply to the more complex interventions of today. These factors include the type of responsible institution, the overall size of the program in terms of number of sites, the monthly number of women attending ANC, and the related logistics challenges associated with these factors. Despite these limitations, we believe that the factors identified as predictors of program performance may help to inform the development of additional public health programs that can help reduce MTCT of HIV in resource-constrained environments such as sub-Saharan African countries.This study identified a number of predictive factors that contribute to uptake of PMTCT coverage in sub-Saharan Africa countries and highlights the challenges and potential benefits of scaling up PMTCT programs in sub-Saharan Africa countries. Additional insight into programmatic factors associated with performance is essential for further expansion and scale-up of PMTCT in resources-constrained countries. Performance may be better for PMTCT programs with a small or medium number of women attended in ANC. While additional research is needed and many public health challenges remain, overall strengthening of heath systems, increasing educational strategies, enhanced counseling, and community support have the potential to improve access to PMTCT services in sub-Saharan Africa.


Immunoglobulin M (IgM) is the largest antibody molecule and is deposited in the glomeruli in a wide variety of both primary and secondary glomerulopathies. The data on its pathogenic role are conflicting till date. A recent study provides evidence for the involvement of natural antibody IgM in fixing and activating complement and causing glomerular injury, proteinuria, and glomerulosclerosis in an animal model. This finding is important in understanding the pathogenesis of the related disorder of IgM nephropathy. IgM nephropathy (IgMN) is a controversial clinicoimmunopathologic entity in the arena of primary glomerulopathies. Recently, many clinicopathological studies have been published, but there is severe lack of information on etiopathogenesis of the condition. A recent study has found that naturally occurring IgM antibody is pathogenic and plays a role in causing damage in animal models and focal segmental glomerulosclerosis (FSGS). This study provides an important stimulus for basic research focused on elucidating the pathophysiology of IgMN.et al.  have been completely observed by the author.None.


Emperor Joseph II: My dear young man, don't take it too hard. Your work is ingenious. It's quality work. And there are simply too many notes, that's all. Just cut a few and it will be perfect.Mozart: Which few did you have in mind, Majesty?Emperor Joseph II: Well, there it is.Amadeus (1984)Quotation from the film Archives and BMJ I am interested in the presentation of numerical data.As a statistical reviewer for digits). The number of decimal places is the number of digits to the right of the decimal point, while the number of significant digits is the number of all digits ignoring the decimal point, and ignoring all leading zeros and some trailing zeros (for a fuller definition see ‘significant figures’ on Wikipedia).A number's precision relates to its decimal places or significant figures .3Take as an example the odds ratio (OR) of 22.68 (95% CI 7.51 to 73.67) comparing beta mimetics with placebo for side effects requiring a change of medication.reporting rules) about rounding numbers, some of which relate to decimal places ”.A second example is the Cochrane Style Guide, which requires risk ratios to be reported to two decimal places.As a third example, p values, it has been suggested, should be rounded to one or two decimal places.1Thus a decimal places rule that ignores significant digits does not work. But equally, and perhaps surprisingly, a significant digits rule that ignores decimal places does not always work either. Reporting risk ratios to three significant digits for example leads to the largest ratio below 1 being reported as 0.999 and the smallest above 1 as 1.01, with three and two decimal places, respectively. This is clearly unsatisfactory as they differ in precision by a factor of ten. In this instance a combination of significant digits and decimal places, the rule of four,The rule of four gives three decimal places for risk ratios from 0.040 to 0.399, two from 0.40 to 3.99 and one from 4.0 to 39.9.11Another example is the reporting of test statistics such as t or F. Specifying one decimal place would permit say t=30.1, where 30 is clearly sufficient as it is so highly significant. Conversely specifying two significant digits would permit t=−0.13, where again the extra precision is irrelevant as it is far from significant. A suitable rule specifies up to one decimal place and up to two significant digits.When comparing group means or percentages in tables, rounding should not blur the differences between them. This is the basis for the Hopkins two digits rule,Recognising the fallibility of decimal places rules means that tables ought not to be restricted to columns of numbers with fixed decimal places, and this adds flexibility when deciding how many decimals to use. For example measures of variability, eg, standard errors (SE)s or CIs, need not be as precise as the effect size, particularly if the CI is wide. A useful trick when formatting table columns is to align the numbers by decimal point, which highlights differences in the number of decimal places. This is particularly useful in columns of risk ratios or p values—see the examples in the table.It is important that any intermediate calculations are carried out to full precision, and that rounding is done only at the reporting stage. This raises the question as to whether the rounded results need extra precision in case they are later included in meta-analyses. But to my mind this confuses two distinct aims—to present the results as accessibly as possible, and to report the results as raw data for meta-analysis. If the two aims are mutually exclusive then the first has to take priority. But in practice they may not be mutually exclusive—where effect sizes are rounded to two or three significant digits the rounding error variance is likely to be small compared with the inter-study variance, making the rounding imprecision relatively unimportant.Fortunately for us, Emperor Joseph did not tell Mozart which notes he should cut, but here researchers have a clear steer as to which digits they can ditch.


In addition, the biosensor also showed good repeatability, reproducibility, anti-interference, and stability. Moreover, the novel Lac based biosensor was successfully used in the trace detection of catechol existing in real water environment. Graphene based 2D nanomaterials have attracted increasing attention in biosensing application due to the outstanding physicochemical properties of graphene. In this work, palladium nanoparticles (Pd) loaded reduced graphene oxide (rGO) hybrid (rGO-Pd) was synthesized through a facile method. Laccase (Lac) was immobilized on rGO-Pd by utilizing the self-polymerization of dopamine, which generated polydopamine (PDA). The PDA-Lac-rGO-Pd nanocomposites were further modified on electrode surface to construct novel biosensing platform. The obtained electrochemical biosensor was applied in the detection of catechol, achieving excellent analytic results. Under the optimum condition, this biosensor possessed a linear range from 0.1  On basis of this, a large number of Lac based biosensors have been prepared for the detection of catechol [To our knowledge, phenolic compounds are hazardous and can cause severe health problems to human beings and animals . These hcatechol –8. LaccaMetal nanoparticles have been widely applied in both chemical sensors and biosensors due to their high surface area, high mechanical strength, ultralight weight, rich electronic properties, and excellent chemical and thermal stability , 10. HowIn this work, Pd nanoparticles loaded rGO (rGO-Pd) was synthesized through a facile method. Lac was immobilized on rGO-Pd by utilizing one-pot Lac-catalyzed oxidation of DA in an aqueous suspension containing Lac, rGO-Pd, and DA. The as-prepared PDA-Lac-rGO-Pd composite was used to modify glass carbon electrode (GCE) to construct novel PDA-Lac-rGO-Pd/GCE biosensor. The biosensor was employed to detect harmful phenolic compound (catechol) in water environment and achieved satisfactory analysis results. This study demonstrates that the PDA-Lac-rGO-Pd/GCE biosensor can serve as an effective water environmental monitor and may find potential applications in other research fields, like biofuel cell, biocatalyst, and so forth.Palladium acetate, expanded graphite powder, dopamine (DA), and catechol were purchased from Aladdin Chemical Reagent Co., Ltd. . Laccase  was obtained from Sigma-Aldrich and used without further purification. Other chemicals were all purchased from the Sinopharm Group Chemical Reagent Co., Ltd. . Acetate buffer solution  was used as a supporting electrolyte. All aqueous solutions were prepared with deionized water (DIW).−1 on a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific).High-resolution transmission electron microscope  was used to observe the morphology of rGO-Pd. Powder D8 Advance X-ray diffraction  and 3D Nanometer Scale Raman PL Microspectrometer  were employed to analyze the chemical components of GO, rGO, and rGO-Pd hybrid. The morphology of PDA-Lac-rGO-Pd composite was observed with a Hitachi SU1510 SEM. Fourier transform infrared (FT-IR) spectra were recorded in the range of 500–4000 cm2 to Pd [Prior to use, GO was prepared from expanded graphite powder by Hummers' method . rGO-Pd 2 to Pd .−1) suspension was prepared with the aid of sonication for ca. 5 min. Then, 1 mL Lac solution (1.0 mg mL−1) and 1.0 mL DA solution (2.8 mg mL−1) were successively added in the rGO-Pd suspension under continuous stirring for 3 h. As shown in 1 mL rGO-Pd  was polished to a mirror-like surface with alumina slurry and washed by DIW. Next, the electrode was immersed in ethanol for 5 min with the aid of ultrasonic to remove the adsorbed alumina slurry. After that, the as-prepared electrode was dried in nitrogen. As shown in Electrochemical experiments were conducted by using a CHI 660E electrochemical workstation . A three-electrode cell was employed for electrochemical tests, which contained a GCE, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode. Before electrochemical experiments, the test solution was bubbled with highly pure nitrogen for 20 min to exclude the influence of oxygen except the amperometric analysis.−1 and 1590 cm−1, which are corresponded to D band and G band, respectively. The D band is relative to the out-of-plane sp3 vibrations of carbons, while the G band can be ascribed to the carbon in-plane sp2 vibrations [ID/IG) reflects the size of sp2-carbon domain and the quantity of defect sites. The ID/IG value of GO is 0.96, while this value for rGO and rGO-Pd increases to 1.34 and 1.36, indicating that rGO and rGO-Pd harbor more defects than GO, which may be attributed to the smaller size of the rGO sheets [2 carbon ribbons during the reaction [X-ray diffraction (XRD) was employed to investigate the chemical components of GO and rGO-Pd. As shown in brations , 31. TheO sheets . It is nreaction . The Ram−1 are related to the in-plane bending vibrations of phenolic groups and CH/CH2 groups, which also can be found in the spectra of PDA-Lac and PDA-Lac-rGO-Pd composites (spectrum (3) and spectrum (4), resp.) [−1 is assigned to NH and OH stretching vibrations in the proteins, which also occurs in curve (c) and curve (d) [−1 can be ascribed to the characteristic absorption peak of amide I (1700–1600 cm−1), and this peak is shifted to ca. 1605 cm−1 in spectra (c) and (d), indicating the strong interaction reaction between Lac and DA [, resp.) . As showurve (d) . The peac and DA . NotablyElectrochemical impedance spectroscopy (EIS) experiments were conducted to investigate the interface resistances of modified electrodes. As shown in µM catechol in 0.1 M pH 5.0 acetate buffer solution by using cyclic voltammetry. Bare GCE (curve (1)) shows a pair of redox peaks, which are located at around 0.47 V and 0.16 V, respectively, indicating the electrochemical redox reaction of catechol on the surface of bare GCE. Compared with bare GCE, the redox peaks of PDA/GCE (curve (2)) are decreased to some extent, which may be caused by the inhibiting effect of PDA layer toward electron transfer. Nevertheless, due to high-efficient catalysis of Lac toward catechol, not only the anodic peak but also the cathodic peak of PDA-Lac/GCE (curve (3)) possesses higher current value than those of bare GCE and PDA/GCE. It is noticeable that the anodic peak current and cathodic peak current of PDA-Lac-rGO/GCE (curve (4)) are increased by around 0.83 µA and 0.77 µA as compared to those of PDA-Lac/GCE, respectively. This demonstrates that addition of rGO accelerates the electron transfer of electrode, further leading to higher response sensitivity of modified electrode to catechol. A pair of redox peaks are observed on PDA-Lac-rGO-Pd/GCE (curve (5)); the anodic peak and cathodic peak are located at about 0.43 V and 0.20 V, respectively. In addition, the anodic peak current and cathodic peak current are 9.78 µA and −7.79 µA, respectively, which are higher than those of PDA-Lac-rGO/GCE, suggesting the higher sensitivity of PDA-Lac-rGO-Pd/GCE. This can be attributed to the larger number of electroactive sites of rGO-Pd offered by Pd nanoparticles and synergetic catalysis of Pd nanoparticles. The whole electrochemical reaction is a quasi-reversible cyclic process and the sensing mechanism of Lac on PDA-Lac-rGO-Pd/GCE is illustrated in µM catechol. The oxidation peaks (anodic peaks) and reduction peaks (cathodic peaks) simultaneously increase with the increment of scan rates. As shown in the inset of To acquire the optimum analytic performance, the test environment including solution pH and applied work potentials was optimized before the chronoamperometry experiment, and the result is shown in µM to 263 µM with a correlation coefficient (R2) of 0.983 (n = 15). The sensitivity is 18.4 µA mM−1 and the detection limit is estimated to be 0.03 µM at a signal-to-noise ratio of 3 (S/N = 3). The steady-state amperometric responses of the PDA-Lac-rGO-Pd/GCE to different concentration of catechol were determined by the successive addition of different volumes of 0.2 mM, 2 mM, and 20 mM catechol into 20 mL pH 5.0 acetate buffer solution with stirring under the optimum conditions; the result is shown in µM), including catechol, hydroquinone, catechin, gallic acid, guaiacol, phenol, and aminophenol. No obvious interference of these interferences to catechol detection can be observed, indicating the anti-interference of PDA-Lac-rGO-Pd/GCE. The repeatability, reproducibility, anti-interference, and stability of PDA-Lac-rGO-Pd/GCE were also investigated. The relative standard deviation (RSD) value for 8 successive measurements is 1.6%, implying good repeatability. To study the reproducibility of biosensor, five PDA-Lac-rGO-Pd/GCE were, respectively, prepared under the same condition and the RSD value is calculated to be 3.2%, which demonstrated that the PDA-Lac-rGO-Pd/GCE possessed acceptable reproducibility. The practical application of the PDA-Lac-rGO-Pd/GCE biosensor was also investigated by using untreated water from Taihu Lake, Wuxi, China. Herein, recovery experiment was used and repeated for five times. The analytical results are shown in In summary, rGO-Pd hybrid was facilely synthesized by a simple method. PDA-Lac-rGO-Pd composite was prepared by a one-pot Lac-catalyzed oxidation of DA in an aqueous suspension containing Lac, rGO-Pd, and DA. A novel biosensor based on the PDA-Lac-rGO-Pd composite was fabricated. Consequently, the biosensor showed excellent biological electrocatalysis toward phenolic pollutant, catechol, with low detection limit, high sensitivity, and wide linear range, as well as good repeatability, reproducibility, anti-interference, and stability. The biosensor was also successfully applied in catechol detection in lake water. This novel composite based on rGO further expands the application of graphene materials in the field of biosensing and paves the way in developing highly sensitive phenolic biosensor.


We genetically modified tobacco mosaic virus (TMV) to surface display a characterized peptide with potent metal ion binding and reducing capacity (MBP TMV), and demonstrate that unlike wild type TMV, this construct can lead to the formation of discrete 10–40 nm gold nanoparticles when mixed with 3 mM potassium tetrachloroaurate. Using a variety of analytical physicochemical approaches it was found that these nanoparticles were crystalline in nature and stable. Given that the MBP TMV can produce metal nanomaterials in the absence of chemical reductants, it may have utility in the green production of metal nanomaterials. N-vinyl pyrrolidone (PVP), and hydroxylamine , poly-xylamine . In contxylamine . Althougxylamine . Moreovexylamine . Therefoxylamine .Recently the metal ion reducing and binding capacities of proteins, peptides, and amino acids have been exploited for the non-hazardous production of nanoparticles from metal salts; reactions which can be carried out at room temperature and neutral pH, with reduced environmental costs and increased biocompatibility . Amino aNicotiana benthamiana was shown to elicit the formation of gold nanoparticles when mixed with a K(AuCl4) precursor. This paper describes a method for production of crystalline, stable free gold nanoparticles using a virus but without the addition of exogenous reductants.While such peptides have excellent potential in nanoparticle synthesis, there are still cost and scalability issues with regard to their large scale production for use in commercial applications. Although peptides can be produced using yeast and bacterial expression systems, plant expression systems have emerged as a cheaper, safer, faster, and more scalable platform for protein and peptide production . For exaPlasmid pSNC004 containing a full-length TMV U1 genome under the control of a T7 promoter and modified in its coat protein sequence at nucleotides 193–198 and 208–213 to contain NgoMIV and BstZ17I sites, respectively, was provided by Dr Sean Chapman . The plasmid was digested with these restriction enzymes and treated with Calf Intestinal Phosphatase  prior to electrophoresis on 1xTBE 1% agarose gels and purification using a Qiagen gel extraction kit . The digested plasmid was then ligated to the MBP primers discussed below.The MBP primers (Au2F and Au2R) below correspond to the SEKLWWGASL sequence which is flanked with incomplete restriction sites to enable downstream ligation to the digested plasmid . These primers which were synthesized by Eurofins  were modified to contain 5′ phosphates.CCGGCTCTGAAAAGCTTTGGTGGGGAGCTTCTCTGTA 3′Au2F 5′ TACAAGAGAAGCTCCCCACCAAAGCTTTTCAGAG 3′Au2R 5′ Escherichia coli and electroporated according to the protocols of g to pellet the bacteria prior to plasmid extraction using a Qiagen Miniprep Kit . The isolated plasmids were sent for sequencing at the James Hutton Institute sequencing service  in order to confirm successful integration of the sequence of interest. Those clones with the correct sequence were subsequently used for generation of infectious TMV transcripts.Primers were annealed by mixing 2 μM of each primer in a 10 mM Tris pH8, 50 mM NaCl, and 1 mM EDTA reaction mix. Reactions were placed in a 96°C heating block for 2 min, after which the heating block was turned off and left until the temperature returned to room temperature. The annealed primer mix was then ligated to the BstZ17I/NgoMIV digested and CIP treated plasmid according to the New England Biolabs  T4 DNA ligase protocols. Two microliters of each ligation was mixed with 50 μl of electrocompetent XL1 blue in vitro transcription, the plasmids were linearized by KpnI digestion and then electrophoresed and purified as described above. The concentration of the digested plasmids was quantitated spectrophotometrically, and 750 ng of each plasmid was then used in a T7 in vitro transcription reaction according to the manufacturer’s protocols . Reactions were diluted 1:50 and abrasive carborundum powder was added, and 10 μl of this solution was rubbed onto the uppermost emergent leaves of N. benthamiana plants at the five leaf stage of development. Plants were grown under 16 h daylength at 23°C. Progression of symptoms was observed over a 16 days period after which material was harvested for virus isolation. The material was ground to a powder using liquid nitrogen and mortar and pestles prior to addition of two parts (w/v) 4°C 0.1 M sodium phosphate buffer pH 7.8, 20 mM EDTA , 0.1% β-mercapthoethanol . After incubation at 4°C for 1 h, the solution was centrifuged at 10,000 g for 20 min at 4°C. Six parts supernatant was mixed with one part chloroform and then centrifuged as before. PEG  and NaCl  were then added to the supernatant at a final concentration of 2 and 1%, respectively, and precipitation was performed overnight at 4°C. After another centrifugation step the pellet was resuspended in 25 mM Tris-HCl buffer (pH 7.8). The virus suspension was purified by a low speed 4°C centrifugation at 2,300 g for 5 min, followed by supernatant ultracentrifugation at 4°C on a 20% sucrose  cushion for 2 h at 32,000 rpm in a SW 41 Beckman Coulter Optima L-80 XP rotor . The pellet was dissolved in 0.01 M Tris-HCl buffer (pH 7.8) and a second round of ultracentrifugation was performed as before but without a sucrose cushion. The virus pellet was resuspended in ultrapure water  and stored on ice at 4°C.Prior to Tobacco mosaic virus particle yield was quantitated spectrophotometrically using A260, A280, and A320 measurements according to N. benthamiana leaves were harvested for western blotting 16 days after inoculation. Approximately 30 mg of leaf material was ground in liquid nitrogen, combined with 100 μl of 2X Laemmli loading buffer and then boiled for 5 min to denature the proteins. After centrifugation at 16,000 rpm, 20 μl was loaded onto 15% SDS-PAGE gels, along with 15 μl of Novex prestained ladder . Gels were electroblotted onto Immobilon-P membrane . The Immobilon-P membrane was blocked by incubating in 1X PBS, 1% BSA and 0.05% Tween, for 1 h. Anti-TMV antibodies raised in rabbits were added to the membranes in blocking solution at a 1/10000 dilution and incubated with shaking at room temperature for 1 h. After washing, an anti-rabbit IgG alkaline phosphatase conjugate was added to the blots at a concentration of 1/1000 in blocking buffer . After incubation at room temperature under shaking conditions the blots were washed and covered with BCIP/NBT . The blots were left to develop for 10 min, after which banding was visible. The developed blots were scanned and saved as jpeg images.Non-infected, wild type (WT) TMV and MBP TMV infected Viruses diluted in sterile filtered distilled water were adsorbed onto carbon-coated copper grids which were examined in a JEOL 1400 transmission electron microscope (TEM) at 80 kV.For immunotrapping, TMV antisera (raised in rabbits) was diluted 1/1000 in 0.7 M Sorenson’s phosphate buffer and 30 μl was applied to carbon coated grids and incubated for 60 min at 37°C. These grids were subsequently washed twice in Sorenson’s by incubating them each time for 10 min on a rotating table. Ground plant material was mixed with buffer to give 1 in 4 and 1 in 40 dilutions (w/v) and filtered through miracloth, prior to overlaying 20 μl of the sample with the antisera coated grids. These were incubated overnight at 4°C prior to washing twice with 50 μl sterile water, treating with 2% uranyl acetate and then air drying before viewing on the TEM.Purified virus was adsorbed onto the surface of freshly cleaved mica for 1 min, after which the solution was carefully removed with filter paper. This substrate was immediately placed on to a drop of double-distilled water (this procedure was repeated twice), and the surface was dried under air flow. This sample preparation method was used to eliminate any remaining salts and minimize any risk of aggregation during drying. AFM analysis was performed on these samples using a Nanoscope IIIA microscope  operating in tapping mode with a typical scan rate of 1 Hz. The measurements were performed in air in tapping mode using sharp silicon cantilevers  with a guaranteed tip radius of 10 nm.Virus preparations were analyzed using DLS and Zeta potential techniques. Measurements were carried out using a Zetasizer Nano ZS device  with a He-Ne laser  as a light source. Sample temperatures were maintained at 0.1°C using a Peltier thermostatting system. Light scattering was measured at an angle of 173°, with subsequent detection and processing of autocorrelation functions being carried out using Dispersion Technology Software (DTS) version 5.10. Polystyrene cells with 10 mm optical paths were used for the DLS experiments, where sample volumes were 1 ml and the concentration of virus preparation was in the range of 0.15–0.3 mg/ml. Zeta potential measurements were carried out using the same sample volume and virus concentration range specified above, except that the samples were decanted into folded capillary cells  for analysis.In a 200 μl reaction, 0.1 mg of purified TMV (WT or MBP TMV) was combined with 3 mM of potassium tetrachloroaurate (prepared in filtered sterile distilled water) and shaken at 100 rpm at 20°C.Gold nanoparticles were characterized using TEM, AFM, absorbance spectroscopy, selected area electron diffraction analysis (SAED), and Dynamic light scattering (DLS). A LEO912 AB OMEGA TEM operating at 100 kV was used in tandem for image capture and SAED. The nanoparticle samples were diluted 1:10 or 1:100 and pipetted onto formvar coated copper grids, which were then air dried prior to insertion into the microscope. AFM measurements were carried out in the same way as for the virus samples.For DLS measurements, samples of gold nanoparticles were loaded into 1 cm cells of the Zetasizer Nano ZS , and measurements were obtained using the He-Ne laser (633 nm). Curves were fitted using DTS version 5.10.UV-visible measurements in the 300–750 nm range were measured in cells with an optical path of 1 cm, using a Beckman DU640 spectrophotometer. The spectra of the viruses were used as a baseline and subtracted from the spectra of a mixture of metal salt/acid and viruses.For XRD, precipitated gold nanoparticles were measured using a DRON-3M X-ray diffractometer with a CuKα X-ray source (aaa = 1.5418 Å) operated at 30 kV and 20 mA. Powder X-ray diffraction data were measured in the 2Θ range from 35 to 45°.EDS measurements were obtained using a LEO 1430-vp (Carl Zeiss) field emission scanning electron microscope equipped with an Oxford energy dispersive detector INCA. The process of sample preparation consisted of drying a 10 μl drop on a silicon substrate. EDX spectra were measured for separate particles to confirm their chemical composition.Nicotiana benthamiana plants inoculated with WT TMV developed the classical symptoms of stunting, leaf malformation and chlorophyll loss at 16 days post inoculation (dpi). In contrast, slightly more severe stunting and malformation was observed in plants inoculated with the TMV modified to display the SEKLWWGASL metal binding peptide (MBP TMV) in the surface exposed loop of the TMV coat protein (the peptide replaced the 66–75 aa region of the coat protein). At 16 dpi, plants were harvested and some material was used for western blot analysis and immunotrapping TEM, while the remainder of the tissue was used for the isolation of virus. Western blot analysis confirmed that the WT and the MBP TMV accumulated to similar levels in plants and that the MBP TMV coat protein was of a higher molecular weight than the WT, indicating successful insertion of the MBP . Immunotrapping TEM using antibodies against TMV illustrated that WT TMV could be successfully detected as distinct rods  in plant material, and also that the MBP TMV existed as aggregated networks of particles of different lengths of up to 300 nm and diameters of 13–18 nm .Subsequently the WT and MBP TMV particles were isolated from the remaining plant material and quantitated using spectrophotometry. It was found that both the WT and MBP TMV could be obtained to high and similar yields (960 mg/kg fresh leaf weight for WT TMV and 1120 mg/kg fresh leaf weight for MBP TMV). These findings are consistent with those reported by Figures 1A,B). As expected, it was found that the unmodified TMV predominantly existed as 300 nm length, 18 nm diameter rods . In contrast the MBP TMV was found as a network of filamentous particles of various lengths of up to 300 nm and widths ranging from 13 to 18 nm . The TEM data was found to correlate well with that obtained using atomic force microscopy . AFM is a high-resolution technique that has been used in studies of biological macromolecules, in particular, for examination of mechanical properties and dimensions of virus particles , whereas those of MBP TMV were 5.9 ± 0.7 nm . The smaller height of the MBP TMV structures may be due to improper assembly and/or instability, which might in consequence be more susceptible to AFM tip compression than the WT virus.The integrity and morphologies of both TMV types was assessed using TEM . Analysis of the hydrodynamic diameter of particles in solution by DLS showed that the unmodified TMV preparation had particles with an average size of about 70 nm, which in general coincides with the DLS data obtained for the other helical plant viruses , which is consistent with the lack of a color change in this reaction. In contrast the MBP TMV morphologies were unchanged by the incubation with gold salt , and had some nanoparticles associated with the network. Interestingly, further TEM analysis revealed that for the MBP TMV reaction large numbers of unattached gold nanoparticles of 10–40 nm size ranges were clearly visible , sizes which are consistent with the broad peaks observed in the UV-visible analysis. Using AFM the heights of the gold nanoparticles were in a size range of 9–33 nm . The nanoparticle sizes visualized under the AFM and TEM also correlate well with the size measurements obtained using DLS approaches, which were 14–30 nm . The samples were subsequently analyzed using SAED, which produced several diffraction ring patterns that depict different phase arrangements and illustrated the crystalline nature of the gold nanoparticles in the samples .We next analyzed the reactions using TEM and AFM and found that incubation of the WT TMV with the potassium tetrachloroaurate led to substantial aggregation of the virus with no obvious metallization or nanoparticle formation . This verifies the formation of gold nanoparticles with crystalline structure. The other minor peaks in Figure 5B can be attributed to X-ray diffraction from precipitated residual salts contained in the solution with the gold nanoparticles. We also measured EDX spectra of the separate gold nanoparticles to confirm their chemical composition and showed that their gold metal atom content was 71.92 ± 2%, with the remaining content predominantly comprising carbon.Powder X-ray diffraction data in the 2Θ range from 35 to 45An important consideration of nanoparticle formulations is their capacity to remain stable and not be prone to aggregation during storage; aspects which can greatly influence the physical and chemical properties and utility of the nanoparticles. Consequently, we measured the zeta potential of the formulations and found that the gold nanoparticles had a potential of -13 ± 0.2 mV, indicating that they have moderate stability and are not so prone to aggregation.Thus, the MBP peptide sequence retains functionality when incorporated into surface exposed loops of the virus and is able to efficiently promote the synthesis of stable crystalline gold nanoparticles without the addition of exogenous chemical reducing agents.In this paper we report that introduction of the MBP motif into a surface exposed region of the TMV CP can lead to the formation of thin networked virus structures that are quite distinct from the typical WT TMV rod morphology. However, this modified virus was not compromised in its ability to replicate and spread long distance and elicit the production of systemic symptoms. It has been reported that the CP of TMV is required for efficient long-distance movement through the plant. Previously amino acid changes have been introduced into key regions of the TMV CP that are required for appropriate self-assembly into the virus structure, and it was found that although some amino acid substitutions disrupted virion structures, they did not compromise virus movement and development of systemic symptoms . This inPreviously It has previously been demonstrated that protein and organic acid mediated synthesis of nanoparticles often leads to direct incorporation of these organic components into the nanoparticle during nucleation and growth phases , or in sin situ to form quickly expanding particles which may engulf TMV surface components and become firmly attached. Our reactions by comparison are much slower (48 h) and employ shaking, which may lower the capacity for nanoparticle engulfment of virus structures and enhance their liberation, especially considering that the peptides we surface displayed have been reported to very weakly associate with nanoparticles . For production of metallized TMV for a multitude of different applications  broaden the types of metal nanoparticles that may be produced during this process, (2) improve the reaction kinetics and (3) develop new approaches for directed metallization of viruses under neutral, non-hazardous, room temperature conditions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Starting from 1700 B.C. in the old world and up to recent times in China there is evidence of earthquake prediction based on unusual metrological phenomena and animal behavior. The review tries to explore the credibility and to pin down the nature of geophysical phenomena involved. It appears that the concept of ancient Greek philosophers in that a dry gas, pneuma is correlated with earthquakes, is relevant. It is not the cause of earthquakes, as originally thought, but may be an accompanying phenomenon and occasional precursor. This would explain unusual animal behavior as well as thermal anomalies detected from satellites.Anomalies in animal behavior and meteorological phenomena before major earthquakes have been reported throughout history. Bio-mimetics or bionics aims at learning disaster anticipation from animals. Since modern science is reluctant to address this problem an effort has been made to track down the knowledge available to ancient natural philosophers. Starting with an archaeologically documented human sacrifice around 1700 B.C. during the Minoan civilization immediately before a large earthquake, which killed the participants, earthquake prediction knowledge throughout antiquity is evaluated. Major practical experience with this phenomenon has been gained from a Chinese earthquake prediction initiative nearly half a century ago. Some quakes, like that of Haicheng, were recognized in advance. However, the destructive Tangshan earthquake was not predicted, which was interpreted as an inherent failure of prediction based on animal phenomena. This is contradicted on the basis of reliable Chinese documentation provided by the responsible earthquake study commission. The Tangshan earthquake was preceded by more than 2,000 reported animal anomalies, some of which were of very dramatic nature. They are discussed here. Any physical phenomenon, which may cause animal unrest, must involve energy turnover before the main earthquake event. The final product, however, of any energy turnover is heat. Satellite based infrared measurements have indeed identified significant thermal anomalies before major earthquakes. One of these cases, occurring during the 2001 Bhuj earthquake in Gujarat, India, is analyzed together with parallel animal anomalies observed in the Gir national park. It is suggested that the time window is identical and that both phenomena have the same geophysical origin. It therefore remains to be demonstrated that energy can be released locally before major earthquake events. It is shown that by considering appropriate geophysical feedback processes, this is possible for large scale energy conversion phenomena within highly non-linear geophysical mechanisms. With satellite monitored infrared anomalies indicating possible epicenters and local animal and environmental observations immediately initiated, the learning experience towards an understanding of the phenomena involved could be accelerated. Predicting earthquakes in any reliable way with the intention to save people and to safeguard potentially dangerous industrial installations is a significant challenge for the future. Even though traditional earthquake precursors from popular tradition have been consistently reported since antiquity and from all major civilizations, and even though more and more compilations and evaluations appear in the literature ,11,12,13At that time, around 1700 B.C., the strong earthquake concerned destroyed the capital Knossos of the Minoan civilisation with its palace, which became known worldwide for its Minotaur and its labyrinth. In fact all stone buildings from that time, on the island of Crete, were essentially destroyed. The palace of Knossos was rebuilt later, however a sanctuary which was situated seven kilometres further south on a hillside below the mountain Jouchtas, the legendary grave of the Cretean god Zeus, in a location named Anemospilia, was not rebuilt. The sanctuary completely collapsed and stayed untouched for 3,700 years. Then the attention of Greek archaeologists was drawn to this place. From there one has a magnificent view over the central territory of the Minoan civilisation. Everything in the small sanctuary, which consisted of three parallel rooms, oriented towards the original capital Knossos, and an entrance room, extended in front, has been conserved, as it was buried by falling wood beams and walls. The central room was reserved for the god-statue and sacrificial donations. Also the room to the right was full of donations, mostly fruit and grain. In the room to the left there was a stone platform, a table, for the preparation of the sacrifice. As well conserved paintings from the Minoan culture clearly show, on such a table during a sacrifice the artery near the throat of a chained bull was cut in order to collect the blood in a sacrificial vessel, especially intended for this purpose, which was then carried to the statue of the god. Four slain humans, the conserved remains of a large statue of a god in the central room, and around 400 pieces of pottery were discovered on the excavation site. Under the careful strokes of brushes by archaeologists and the critical eyes of criminologists a dramatic scene was brought back to life, which documented the intentions of the people concerned, immediately before the terrible earthquake. All this was later documented in a very detailed account published in the Magazine of the National Geographic Society .The temple servant, who was supposed to carry the sacrificial chalice with the blood to the central room of the sanctuary was slain on the way there and the blood-chalice was crushed into 105 pieces (which were carefully reassembled by the archaeologists).In the side room, close to the stone-made preparation table, the slain high priest was found with an exceptionally rich decorated long sacrificial knife. Beside him a priestess was smashed to the ground. The surprise was that, on the stone-table no chained bull was found, but a confined human, an approximately 18 year old young man. The immobility of his limbs was deducible from his position. His body was, when the ceiling of the sanctuary hit him, in part already free of blood. This was clearly determined by the criminologists on the basis of the colour of the bones. In the last few minutes before this severe earthquake struck, the high priest apparently tried to stop the impending calamity by a human sacrifice. Or was the coincidence with the earthquake just chance?For the Minoan culture a human sacrifice was a possible, but not at all a common ritual. The story of the Minotaur, a creature half bull, half man, living in the Labyrinth, is an important example from the far past. The city of Athens had every year to send seven youngsters and seven girls for sacrifice, until the hero Theseus, supported by the daughter of the King Minos, Ariadne with her wool clue, killed the Minotaur. In Knossos another example of human sacrifice was found. In a building dating back to 1500 B.C., which had been destroyed by fire, ritual vessels were found, which suggested that it was a sanctuary. Here the bones of two children were found with cuts, which suggested that they were slaughtered like the bulls and that the flesh had then been taken from their bones.Though the ritual may be rare and reserved for very special occasions, the earthquake in 1700 B.C. has obviously immobilized the ritual of a human sacrifice.What can be learned from such a coincidence with the very strong, 3,700 year old earthquake?First it would be entirely unrealistic to believe, that the very rare case of a human sacrifice would have had another purpose rather than the impending earthquake. The chance of such a coincidence would have been far too small. Furthermore, it happened not in a town centre but in a remote hill sanctuary, which was obviously designed to deal with natural phenomena. The population of the seven km distant town of Knossos was apparently warned, because practically no victims of this severe earthquake were found in ruins elsewhere. Or were they removed later?Assuming, that there was a relation between sacrifice and earthquake, the high priest must have observed something, which convinced him that a natural disaster of this type was imminent. Smaller foreshocks could not have been the reason. In the Aegean Sea there are far too many small earthquakes, without a large one following, to justify a human sacrifice. There must have been another phenomenon, which convinced the priest of a large impending earthquake. It must have been apparent hours before, because it is practically impossible to prepare a human sacrifice within a few minutes. Already the time period needed to take such a serious decision must have been quite long. Then comes the problem of finding a victim and transporting it to the temple. Who was the 18 year old male victim? Was it a slave or was it a voluntary victim from the temple community, who was chained to the sacrificial platform? Something must have escalated in nature to justify such a desperate step as a last resort.This archaeological evidence of natural disaster anticipation does not stand alone in Greek antiquity. In 1650 B.C. the volcano of Santorin erupted and destroyed this and neighbouring islands. When excavating the ash- and lava-buried Minoan settlements archaeologists expected human remains similar to those found in Pompeii. The fact is that no human victims were found, even though approximately 30,000 people are believed to have lived on Santorin island. Even the amphorae used for food storage were found to be empty. Clearly people had all left safely with their essential belongings before the disaster. There is written evidence for earthquake anticipation during antiquity: The writer Cicero reports on earthquake prediction, which occurred in the 6th century B.C. in Sparta. One day the natural philosopher Anaximander told the people of Lakädemonia that an earthquake would arrive and asked them to leave their houses and the town and to camp during the night outside in full armament. His recommendation was followed, the entire town collapsed and a large piece of rock came down from the mountain of Taygetos . The natIn fact, earthquake precursors were known and described during antiquity. The Roman natural scientist Pliny the Elder cites four earthquake precursors: foreshocks, turbid water, exited animals, a strange fog . The firSince the Minoan sanctuary, which was destroyed by the earthquake 1700 B.C., was situated on the northern slope of a mountain, Jouchtas, associated with the most powerful god Zeus (later named Jupiter by the Romans), the sacrificial ceremonies performed in this sanctuary should have been related to worship associated with Zeus or one of his descendents. According to Greek mythology Zeus had god like or semi-god like children with approximately 37 women. Among them, there is only one god, who is immediately interesting for our investigation, since he alone is responsible for the powers of nature and of nature’s fertility. It is god Dionysos or, as the Romans later knew him, god Bacchus. Mythology, according to the most prominent tradition, tells that Zeus (Jupiter) had his son Dionysos (Bacchus) with Semele, the daughter of the Theban King Kadmos. He was educated by the nymphs of Nysa and later by the strange, bald fellow Silen. Now it becomes interesting: when grown up Dionysos (Bacchus) married the daughter Ariadne of King Minos from Knossos in Crete. Coincidentally, Ariadne was the woman who helped Theseus to defeat the terrible Minotaur by providing the wool clue and a sacred sword. This means, that the god Dionysos (Bacchus) is actually linked with Knossos. It may therefore indeed be that the Dionysos/Bacchus cult, for which we have later written and pictured records, is actually a late continuation of the discussed ancient ceremonial practice involving a human sacrifice in 1700 B.C. A first strong support comes from Roman sources, indeed associating the Bacchus cult with ritual human sacrifices. Because of these human sacrifices and because of ecstatic ceremonies and orgies during special festivities , the senate of Rome in 186 B.C. severely restricted these activities . Later the Bacchus cult became therefore mostly associated with wine consuming excesses and the pleasures of life. Such an attribute gave it a superficial and easy-going appearance. This may not always have been the case. Ceremonies leading to ecstasy are known from many natural tribes and are typically associated with now quite well understood shamanic practices. It has always been a practice aimed at assuming contact with the other world, often in an effort to recognise impending danger and to control the future. There are many figurative representations of Bacchus associated with an easy-going way of life. I found only one showing him as the lord of natural forces . It is aHowever, remembering that Bacchus is the traditional Lord of natural forces, and since a volcano reflects a real earthquake danger, we may look at this scene and especially at the animals in an entirely different way. Pliny the Elder, who coincidentally was killed during the same volcanic eruption which eradicated Pompeii, and which conserved this beautiful painting (which Pliny the Elder may have known), reported, as already mentioned, four phenomena, which could precede an earthquake . One wasThe painting of Bacchus and Vesuvius  should tFor our initiative with respect to ancient earthquake knowledge the result is that one strategy of earthquake anticipation known within the Bacchus cult was apparently the observation of anomalous animal behaviour, as depicted in Another interesting fact is that the Dionysos/Bacchus cult has always been associated with processions and theatre-like presentations in honour of the god . Processions also played a non-negligible role in connection with earthquake danger. When wolves penetrated to the Roman Capitol in the year 458 B.C. an expiation ceremony was ordered. People had to leave their houses and to join a procession with decorated cattle. By the way, such a procession is also depicted on the above mentioned marble bas relief from Pompei . The town had to be circumvented three times by the procession and at the end of this time consuming ceremony, the animals were sacrificed to the god. If an earthquake were to have hit, most people would have been in the open air and would have survived. Earthquake prediction attempts were hidden in religious practices, against which nobody could argue, and if the earthquake did not come, it was god`s will or the result of a successful ceremony aimed at avoiding it. When extrapolating back to the initially described human sacrifice preceding a large earthquake in 1700 B.C., we may consequently reach the following impression of what happened during the earthquake tragedy in ancient Crete: The priests serving the god responsible for natural disasters recognized atmospheric and animal anomalies and decided to act. While the numerous population of the capital was engaged in a longer lasting procession in the open air, the chief priest and his aids were on duty in the temple near the statue of god, performing a human sacrifice to fend off the impending earthquake. They were killed by the natural disaster. The second strategy, which should be followed to find an explanation for the anticipation of an earthquake in 1700 B.C. is to follow and analyse recorded ancient knowledge on earthquakes. Starting from the 7th century B.C., over a dozen antique philosophers or scientists theorized about earthquakes: among them Thales of Milet (appr. 650–560 A.D.), Anaximenes , Anaxagoras (appr. 500–428 B.C.), Demokritos (born around 460 B.C.), Antiphon (490–411 B.C.), Aristotle (384–322 B.C.), Theophrastos (371–287 B.C.), Straton of Lampsakos (320–270 B.C.), Poseidonios (135–51 B.C.), Pliny the Elder(24–79 A.D.), Seneca (55 B.C.–40 A.D.), Ammianus Marcellinus . They tried to find natural reasons and forces for the occurrence of earthquakes, even though religion suggested angry and punishing gods as the origin of such disasters. There was no contradiction: natural forces were also considered to be controlled by a god such as Dionysos/Bacchus. It is reasonable to assume that these early natural philosophers based their theories and their knowledge on earthquake precursors, on then commonly discussed and popularly recognized real observations of phenomena associated with earthquakes. Due to the low population density and the simple way of life, the environment during antiquity was still largely unpolluted, so that meteorological and biological earthquake precursors could better be distinguished. People also had more time to observe nature.Different explanations have been advanced during antiquity to explain why the earth is shaking. However the most important, widely distributed and persistent idea was that gases, called “pneuma” were involved in the build up and generation of earthquakes. They may have entered the earth from outside, or may have formed inside. There must have been practical evidence for these gases. Something must have been observed that suggested gases were escaping before and during earthquakes.Anaxagoras (according to Aristotle ), in theThe strange cloud or fog, which appears before the earthquake, and is described by Aristotle, has also received attention during a later historical event and Pliny the Elder explicitly mentions it as one of four possible earthquake precursors . The RomRemarkably, and typical of all antique understanding of earthquakes, Theophrastus,  discThe pneuma theory of earthquakes received further studies from Straton of Lampsakos, the teacher of Ptolemaios II, from Poseidonios, (as reported by Seneca) and from Ammianus Marcellinus, in his “Res gestae” ,23.Also during all the Middle Ages the “pneuma” earthquake theory of escaping gases continued to be respected as a reasonable explanation, for example by Albertus Magnus or Thomas Aquino. We are thus dealing with an ancient scientific concept on earthquake phenomena that has amazingly survived at least during two millennia. With the development of modern science and the establishment of the geo-tectonic concepts of earthquake events, the “pneuma”-concept of gases escaping from the ground prior to and during earthquakes became an imaginative invention of scientifically naive early natural philosophers. The now accepted and verified notion that escaping gases are not responsible for earthquakes however, obscured the possibility that they still could participate in the earthquake event as precursors or accompanying phenomena. For this reason nobody really cared to look with modern scientific tools for escaping “pneuma”-gases prior to or during earthquakes. The age-old experience and knowledge of early scientists in relation to a possible side phenomenon of earthquakes was simply ignored. The aim of this paper is to show that it may also be relevant for understanding unusual animal behaviour before earthquakes. What ancient natural scientists really could have experienced during their lifetime or could have communicated as ancient knowledge, or what the high priest from the discussed earthquake disaster on Crete in 1700 B.C. could have observed, may best be illustrated with a report from a more recent earthquake event on another Mediterranean island: During a visit of participants of the 3rd International Conference on Earthquake Precursors (ICEP), Naples , to the site of the town Gibellina in Sicily, which had been completely destroyed by an earthquake in January 1968, the author asked eyewitnesses whether precursors had been observed and he got surprising information from a quite reliable source . The quaIf we tentatively take these singular observations of pre-earthquake phenomena associated with the Gibbelina-quake for granted and compare them with what the ancient naturalists were reporting, we immediately realize an astonishing similarity. A metrological situation arises, which resembles a smog laden heat island around a densely populated modern city. There is the strange fog, which may dim the sun light. And there is the long stretched thin cloud on the horizon, which, according to Aristotle, “is visible during the day or shortly after sunset”. Aristotle also compares it with a surf-line. This suggests that it is seen close to the horizon, like the haze produced by smog. The narrow smog pattern above the horizon, which assumes a reddish appearance during sunset is nowadays a well known phenomenon seen around large urban centres. The “pneuma” gas which was seen escaping from the earth was considered to be warm and dry. Such a condition has frequently been reported as typical “earthquake weather” from many earthquake regions of the world . SensitiThe appearance of gases and the accompanying alteration of the earth near atmosphere together with an approaching earthquake receive strong support from popular tradition. An analysis of 78 reported documents on relevant earthquake precursors from different civilizations  allowed Such a phenomenon would be imaginable as a consequence of pressure changes underground. It could induce a change of chemical equilibrium, of pressure induced electrical phenomena , or of oAnimals, depending on the species, are known to respond to many physical and chemical signals in a much more sensitive way than humans. They can be sensitive to very different signals. They may respond to different signals before earthquakes. Most comparative information on animal behaviour before earthquakes was compiled during the Chinese initiative for earthquake prediction on the basis of animal and environmental precursors, which occurred during the sixties and seventies of the past century.The Chinese initiative for earthquake prediction, which took place fifty years ago following a political decision, was based on a broad cooperation of peasants and agricultural and farm workers, who were informed and trained with illustrated leaflets and fans on how to judge unusual animal behaviour before earthquakes . UnusualIt has been argued  that earThe Chinese authorities, thus proceeding in an entirely empirical way, used to give an alarm, when the incoming reports on animal and environmental anomalies escalated. They have claimed to have predicted up to 10 earthquakes, the best known being the earthquake of Haicheng on 4 February 1976. It was a magnitude 7.3 earthquake, shortly ahead of which the population was evacuated from their houses into the snowy open landscape. Characteristic longer term indications for the arrival of this earthquake were snakes which left the underground to freeze on the icy surface. I myself have seen many of these amateur photographs, when I visited the Institute of Biophysics in Beijing in 1988. Abnormal animal behaviour in the Haicheng area was observed in 800 cases and concerned 30 animal species (cited in ). Another large destructive earthquake, five months later, on 27 July 1976, the magnitude 7.8 Tangshan earthquake, however has not publicly been anticipated. It occurred at 3.42 A.M. and caused at least 240,000 deaths, according to other Chinese sources 655,000. Mainly because the prediction did not work and due to the internationalization of Chinese earthquake research the interest in systematically studying abnormal animal behaviour for earthquake prediction had ceased. In fact, the failure to predict the Tangshan earthquake was taken as demonstration of the incapacity of a prediction strategy based on mass participation in recognizing animal and other traditional precursor phenomena . The siAccording to the information obtained by the author in 1988 in the Institute of Biophysics, Chinese Academy of Science, in Beijing the failure of Tangshan earthquake prediction was to a large extent caused by political turbulences during July 1976 between the left wing “gang of four” and the pragmatic wing of the communist party around the termination of the cultural revolution. Personalities responsible for giving earthquake alarm were in part simply removed from their positions and could not act. Other opinions, however, also exist and there are detailed reports on the earthquake experience in China which could be considered as a reference e.g., ,31). . 31]). Two days after the disastrous Tangshan quake researchers from 16 Chinese research institutions formed a research group and started studying many aspects of the earthquake including animal precursors. They published a 459 page thick report on “The Tangshan earthquake of 1976” during 1982 . ChapterOn the basis of well defined standards and geographical patterns reported animal phenomena from 412 people’s communes spread over 60 districts were studied during this initiative of the Chinese earthquake study commission. The working force consisted of members of the biological and chemical seminary of Nankai University, the Pedagogical University of Tianjin and of the Geological Research Institute of Yejin. Most cases of abnormal animal behaviour concerned 30 animal species with abundant presence in the region. As many as 2,202 cases of abnormal animal behaviour were identified in the region affected by the earthquake. However, all together, nearly 70 animal species were concerned. About 80% of the reports studied were considered reliable with respect to time and location and were directly observed by identified eyewitnesses. The rest were second hand reports. In 45 cases observers of animal anomalies expected an impending earthquake and notified authorities. The animal precursor was reported before the quake. In the majority of cases the anomalies observed gained importance only after the quake. For example, this happened in the case of a woman from the village of Daodi, in the district of Fengnan. Before she died of her injuries she said that on the evening before the quake the children had insisted that mice were behaving so strangely that an earthquake may be imminent. The woman considered this as nonsense and did not react. Unfortunately the quake arrived and the children did not survive. The general abnormal animal behaviour observed prior to the Tangshan earthquake matched that observed with other major earthquakes in China. Two categories could be distinguished. One is a reaction of fear in animals, the other a reaction of melancholy. Horses, cattle, and donkeys were frightened, refused to enter their stable, and did not eat. Also pigs and sheep refused to enter their pens, and if already in them, tried to break out. Dogs barked in an exited way, ran around, sniffled on the ground, and evacuated pups from the houses, carrying them in their mouths. Also cats evacuated their kittens, were restless, made a noise, and woke up their owners. Rabbits refused to eat, made a noise, and attempted to evacuate their pens. Chickens refused to enter their stables, and jumped and flew up to high branches of trees. In the pens they were frightened as if a marten were around. Ducks and geese avoided going into water or fled from it, did not eat, and refused to enter pens. Pigeons avoided their loft, and flew frightened in swarms. Fish circulated in shoals close to the surface, and jumped from the water, as well as from an aquarium. Mice formed swarms either to leave the area or to rest motionless. They were no longer frightened of man. Before the Tangshan quake there was also some very unusual and dramatic animal behaviour. In the district of Wuning more than 20 eye witnesses observed over 100 fire-weasels running towards a village centre. Later on 10 of them circulated aimlessly around a walnut tree. Five of these were slain by local people. One occurrence also reported before the earthquake was the sighting of approximately 100 mice, who rested quietly on the ground within an industrial plant in Jixian. From the districts of Qian’an and Ninghe large swarms of dragon flies were reported. One extended for 100 meters. From the apiaries of the district of Qianxi and the people’s commune Miaolingtou nearly all of more than 100 and 30 beehives, respectively, were evacuated by the bees before the earthquake. They all took off in a given direction. Near the harbour town Tingbo a large oil tanker became literally covered by dragon flies, butterflies, grasshoppers, crickets and cicadas. They could be touched by hand.Around the rice fields of the village of Hangu and also in the district of Qianxi the intensive quaking of many frogs was a regular evening spectacle. However during the night preceding the earthquake no tone was heard. An effort, by a local person, to capture frogs that night failed. The frogs seemed to have interrupted their activity and to have moved away. The day before the quake several hundred snakes were observed in the district of Tianjin rolled up and calm near a water pond. When startled, the normally shy snakes entered first the water, but then immediately returned to the ground assuming the same position. Near the city of Tianjin and near the people’s commune Xiyingmen large swarms of bats were observed during the bright day preceding the earthquake. The breeding station for sable in Dashantou had a stock of 415 animals, when, two hours before the earthquake, the keeper was alarmed by excessive noise. He suspected that an animal had broken out, but he discovered nothing special besides unusually excited animals. It was found that the unusual animal behaviour before the Tangshan earthquake was concentrated on the areas where the quake was strongest , and where the critical fault lines were concentrated. When, within the region with more than seven on the Richter scale, only areas within 5 km from the fracture lines are evaluated, an interesting observation can be made. The density of abnormal animal behaviour there is found to be five times higher than in the rest of the affected central area. This observation, the correlation of animal anomalies with fault lines coincides with similar ones from other Chinese earthquakes, for example that of Haicheng.The abnormal animal behaviour around the Tangshan earthquake started as , right. The above analysis of animal unrest before the Tangshan earthquake shows that the animal phenomenon was relevant and could have been useful for earthquake prediction  like in the case of Haicheng. In fact, in one of the counties hit by the Tangshan quake the earthquake alert system actually did work. It was in Quinglong county. This example shows an exception to the apparently politically motivated turbulence during the failed Tangshan earthquake prediction efforts.The UN global program for the integration of public administration and the science of disasters conducted a detailed study of the pre-earthquake strategy of Qinglong county ,33. Like other counties of the region later affected by the Tangshan earthquake, Qinglong had started a massive education campaign in anticipation of a major earthquake. A total of 70,000 books and 14,000 posters were distributed and 120 slide shows presented. Sixteen county-, 42 commune-, and 442 village-level observation stations were established and staffed. The lay stations watched for changes in water properties, animal behaviour, geo-electricity and geo-magnetism. Two weeks before the Tangshan earthquake the young administrator Wang Chunqing participated in a conference organized by the State Seismological Bureau, during which warning of an imminent earthquake was given. The county acted by relocating school classes and moving teaching and business activities outdoors. An around-the-clock earthquake watch was organised. While the instrument group (electric and magnetic measurements) did not see any unusual changes nocturnal animals like weasels and rats were observed to move in full daylight. Also water springs became muddy. By 26 July, one day before the disaster, many of Qinglong’s 470,000 residents had moved into tents, the rest slept with open doors and windows as a strategy for fast escape. Even though more than 180,000 buildings collapsed, only one person died and that was of a heart attack.The tragic Tangshan earthquake experience does not disprove abnormal animal behaviour as potential earthquake precursors. In contrast, it adds valuable information.If earthquakes are preceded by precursor phenomena such as animal unrest, some energy is needed to generate them. Since, during energy conversion, energy is finally converted into heat, unusual heat generation should consequently precede major earthquakes.Past earthquakes can be analysed for pre-earthquake thermal anomalies using recorded satellite data . This has been done for a series of more recent earthquakes, such as Bhuj , Boumerdes , and Bam  but also for several additional earthquakes, larger than magnitude six on the Richter scale, in China , Afghanistan  and Pakistan  ,45,46,47What is already known about satellite observed infrared anomalies? The observed anomalies suggest a rise of temperature by a few degrees and up to 10 degrees. This is under the hypothesis that the surface did not change its radiation properties (emissivity). The Bhuj earthquake of 26 January 2001 in Gujarat, India, was, for example, based on such an assumption, preceded by a 4 °C or 5–6 °C temperature anomaly, according to different evaluations ,45 just The reported temperature anomalies ,48,49,50What relation do these modern indications on exhalation of gases before earthquakes have with the “pneuma” concept of Greek and Medieval philosophers and to reported animal earthquake precursors from popular tradition?In 1700 B.C., a severe earthquake levelled a small temple overlooking the area of the Minoian cultural centre of Knossos on the island of Crete. Careful archaeological research revealed, that the persons slain had just performed a human sacrifice, rare for the Minoian civilisation. The priest in charge had obviously tried to avert the natural disaster. Theoretically such a conclusion may be debated, but the rareness of human sacrifices in Minoian culture and other discussed circumstances are conducive to the later Dionysos/Bacchus cult, which indeed concentrated on disaster anticipation. What were the precursors recognised? In order to answer this question two strategies were followed. One was to track down the supposed following up of religious traditions of the Dionysos/Bacchus cult, which centred around the worship and control of natural forces. The second was to understand on what natural phenomena the Greek philosophers were basing their understanding of earthquakes. The conclusion reached is that the priest was judging atmospheric anomalies, related to a gas escaping from underground and to the formation of local heat islands, with obvious consequences for animal behaviour. Modern satellite observations of heat anomalies preceding larger earthquakes appear to identify temperature anomalies in relation to gas emission, which appear to be consistent with the reported ancient observations. That the Minoan culture had experience with disaster anticipation is also supported by the complete absence of victims, in the excavated settlements, of the disastrous volcanic eruption of the Santorin volcano around 1650 B.C. However, the kind of precursors observed is not known. Searching for the religious background and heritage of the earthquake averting ritual, we have identified the Bacchus cult in Rome with its ritual human sacrifices. They were aimed at calming down the god who controls natural forces. A painting of Bacchus in front of the Vesuvius  was reinIt has been shown that modern satellite observations of infrared anomalies before major earthquakes suggest the same conclusion, that gases escape and generate local greenhouse effects. However they need, of course, to be better evaluated and matched with meteorological evidence. All together the assumption, that the satellite detected thermal anomalies producing escaping greenhouse gases are related to the ancient “pneuma” concept of escaping gases provides a reasonable working hypothesis, which could be tested with appropriate experiments.It remains now to be tested whether the cross linking between atmospheric changes and animal anomalies is consistent.Experience up to now with thermal anomalies before earthquakes suggests that they appear in the range of 10 days, one week or a few days or hours before the earthquake, increase towards the disaster and disappear within one day after. Reported animal anomalies cover approximately the same time period. A well known report from the 4th century B.C., for example, cites that five days before the destruction and drowning of the Greek harbour town of Helice, all animals, especially rats, snakes, weasels, millipedes and worms started to leave the city . From 78In order to find out whether temperature anomalies and animal anomalies coincide it was attempted to select an earthquake, for which thermal anomalies were reported and to look for coinciding animal anomalies, which have been reliably documented. We selected the Abruzzo earthquake of 2009 for which infrared anomalies  are docuSince the documented earthquake related temperature anomaly, apparently caused by a greenhouse effect generated by gases escaping from underground, affected this Indian national forest, there is obviously a direct connection with the anomalous animal behaviour. Animals recognise such signals from the underground, which indicate a clearly changing environment, which is frightening them. Behavioural scientists know that the erect tails of cats signal aggression and threat. With the erection of the tail they apparently attempt to increase their size. In an earlier study  we gave The early Indian scientist Varahamihira (505–587 A.D.), by the way, already knew about abnormal animal behaviour and interestingly also about a strange weather anomaly preceding an earthquake in his homeland. In his work Brihat Samhita (The Great Compilation) he speaks of unusual cloud formation a week before its occurrence. It may be the same phenomenon recognized now as infrared anomaly from satellites.Most earthquake researchers and geologists today are still very reluctant to accept phenomena from the popular tradition as credible. The consequence is that they remain almost unexplored. One reason for the reluctance is that they understand the earthquake as a movement of plates, and cannot understand why there should be a significant energy release before. We have dealt with this question in another study  and showOur hypothetical conclusion is that priests and early scientists from Greek antiquity were attentive observers of weather phenomena and had a clear concept of the atmosphere under normal conditions . Occasionally, and under favourable geological conditions they saw smog rising from the underground, which cumulated in earthquake events. They based the “pneuma” theory of escaping gases on such observations and had thus also an explanation for unusual animal behaviour. They attempted to avert earthquakes through sacrifices, since they considered them a punishment from the gods. Therefore observation of earthquake precursors became important and was the task of priests.Modern seismology is essentially based on sensitive mechanical instrumentation and the expectation that any mechanical soil movement during the quake can be monitored from a considerable distance. The accepted mechanism for earthquake generation gives little room for localized gas emission or nervously behaving animals. Satellite based infrared emission anomalies may eventually re-confirm the “pneuma”-theory of ancient natural philosophers, with the restriction, of course, that the “pneuma”-gas is not causing the earthquake but simply preceding and accompanying it. With such a concept, unusually behaving animals before earthquakes may become an understandable phenomenon, provided substantial experiments are performed, along the line discussed in the following section. etc.) A narrow time window of one to several days for intensive animal and geophysical studies would become available which gradually could lead to accumulation of valuable information. Funding, for such initiatives, should be prepared in advance. Biologists may then find out how animals react and what they sense and physicists may support them by analysing gas and aerosol composition in such developing earthquake epicentres. New knowledge will become available on earthquake phenomena and we may also understand more about antique earthquake concepts. This may, hopefully, over the years increase our understanding of earthquake precursors to a standard sufficient for averting danger for people and critical industrial installations. In any case we will then finally know whether the two (of four) earthquake precursors, animal and fog anomalies, which Pliny the Elder described nearly two thousand years ago, and which modern science today still ignores, are based on actual natural observations.It is hoped that our analysis on attempted earthquake forecasting during a period of more than three millennia, throughout antiquity, as well as in more modern times during the Chinese earthquake prediction initiative, will motivate some scientists to look closer into the relationship between thermal anomalies, animal behaviour and earthquakes. A first step, besides intensifying and refining satellite observations, could be to follow the idea of ancient scientists such as Aristotle, Poseidonios or Theophrastus and to include earthquake studies in the field of metrological science. Nowadays an immense number of meteorological data from a wide area is daily evaluated by computer programs and used to predict the weather in specific locations. This procedure is especially requested for the prediction of unusual weather conditions like storms. Earthquakes appear, at least in certain cases, also to generate unusual weather conditions. When predicting temperature and weather data for specific areas known for earthquakes, meteorologists should subsequently and systematically check whether their prediction was correct and what their error was. They should specifically look into temperature and weather anomalies, which they cannot explain on the basis of meteorological experience. Most work could be done on an automatic basis using appropriate computer programs analysing the vast quantity of meteorological data which is continuously generated. This way, and including improved satellite data whenever possible (during clear weather), temperature and weather anomalies coinciding with earthquakes could be more accurately identified. If they were to be recognized in time before the earthquake, experts could be informed and activated to start immediately observing and monitoring animals in zoos, on farms and in the wild. Suitable animal observation strategies with high statistical value should be prepared (e.g., variations in egg production, sound in bee hives, permanence of animals in pens, swarm formation in wild species