Frecuencia de factores asociados a ideación suicida en pacientes con fibromialgia en dos hospitales de Lambayeque 2015

Objetivo: determinar la frecuencia de los factores asociados a ideación suicida en pacientes con fibromialgia en dos hospitales de Lambayeque, 2015. Material y Métodos: estudio descriptivo transversal con análisis exploratorio. Se entrevistó a pacientes con fibromialgia de consultorios externos de los hospitales Regional Lambayeque (Hospital 1) y Almanzor Aguinaga Asenjo de Chiclayo (Hospital 2). Se aplicaron 5 cuestionarios: la escala del centro de estudios epidemiológicos para depresión, escala de Zung para ansiedad, índice de calidad de sueño de Pittsburgh, escala de valoración análoga del dolor y la escala de ideación suicida de Beck, adaptada a población Lambayecana. Se realizó análisis univariado y bivariado exploratorio. Resultados: Se entrevistaron 125 pacientes, 61 del hospital 1 y 64 del Hospital 2; 87%, fueron mujeres. Hubieron 73,6% malos dormidores, 68% tuvieron depresión, 22,4% ansiedad, 24,8% dolor corporal difuso moderado-severo y 30,4% ideación suicida, 27,9% y 32,8% en el primer y segundo hospital, respectivamente. En los pacientes con Ideación suicida la frecuencia fue: mala calidad del sueño 81,6% (p=0,1), depresión 71%(p=0,5), ansiedad 42% (0,001) y dolor corporal difuso (DCD) de moderado a severo 26,3% (p=0,005). En el multivariado, ansiedad, rp= 1,4 IC95%= 1,14-1,7, p= 0,006 y ser mal dormidor, rp= 1,2, IC95%= 1,0-1,4, p=0,01 se asociaron a Ideación suicida. Conclusiones: la frecuencia de ideación suicida es elevada en estos pacientes. El factor más frecuente fue mala calidad de sueño. Ansiedad y ser mal dormidor se asociaron a ideación suicida.Tesi

FKBP12-4 is required for complete hyphal growth.

<p><b>(A)</b> Growth of <i>A</i>. <i>fumigatus</i> (10⁴ conidia) on GMM at 37°C for 5 days, with colony diameter measured every 24 hours, revealed no statistically significant difference in growth among Δ<i>fkbp12-1</i>, Δ<i>fkbp12-2</i>, Δ<i>fkbp12-1</i>Δ<i>fkbp12-2</i>, Δ<i>fkbp12-3</i> and wild-type strains. Δ<i>fkbp12-4</i> demonstrated reduced growth rate across all 5 days (p = 0.0161). <b>(B)</b> After the 5 day growth period, Δ<i>fkbp12-4</i> demonstrated reduced growth compared to wild-type strain but no other obvious phenotypic abnormalities were noted.</p

Phylogenetic analysis of FKBP12 proteins and multiple sequence alignment comparing residues important for FK506-FKBP12 binding.

<p><b>(A)</b> Phylogram showing orthologous FKBP12 proteins from Human (HsFKBP12), <i>S</i>. <i>cerevisiae</i> (ScFKBP12), <i>C</i>. <i>neoformans</i> (CnFKBP12), <i>M</i>. <i>circinelloides</i> (McFKBP12) and <i>A</i>. <i>fumigatus</i> (AfFKBP12-1, AfFKBP12-2, AfFKBP12-3 and AfFKBP12-4). Phylogenic analysis was performed using the respective amino acid sequences aligned with MUSCLE (v3.8.31) implemented in the PhyML program (v3.1/3.0 aLRT). Graphical representation of the phylogenetic tree was performed with TreeDyn (v198.3). <i>A</i>. <i>fumigatus</i> FKBP12 proteins are designated as AfFKBP12-1, AfFKBP12-2, AfFKBP12-3 and AfFKBP12-4, respectively. <b>(B)</b> 14 residues are known to be important for binding FKBP12 to FK506. Residues distinct from human FKBP12 are highlighted in a different color for each <i>A</i>. <i>fumigatus</i> FKBP12 and for each species-specific FKBP12 homolog.</p

Construction of the FKBP12 deletion strains.

<p><b>(A) Construction of Δ<i>fkbp12-1</i> strain</b>. In the Δ<i>fkbp12-1</i> strain, wild-type <i>A</i>. <i>fumigatus fkbp12-1</i> (637 bp) was replaced by the 3.0 kb <i>A</i>. <i>parasiticus pyrG</i> gene. Three of the five strains validated by PCR were then selected for Southern analyses. SalI-digested genomic DNA was probed with the 646 bp probe of the downstream flanking sequence to confirm homologous recombination. Two of the three tested strains demonstrated the expected ~3.3 kb length, which is contrasted with the WT length of ~1.1 kb. Gel used for Southern analysis was 1% agarose. <b>(B) Construction of Δ<i>fkbp12-2</i> strain.</b> In the Δ<i>fkbp12-2</i> strain, wild-type <i>A</i>. <i>fumigatus fkbp12-2</i> (709 bp) was replaced with the 3.0 kb <i>A</i>. <i>parasiticus pyrG</i> gene. The strain validated by PCR was then used for Southern analyses. BamHI-digested genomic DNA was probed with the 733 bp probe of the downstream flanking sequence to confirm homologous recombination. The strain demonstrated the expected ~1.1 kb length in contrast with the ~2 kb length in the wild-type strain. Gel used for Southern analysis was 1.5% agarose. <b>(C) Construction of Δ<i>fkbp12-3</i> strain.</b> In the Δ<i>fkbp12-3</i> strain, wild-type <i>A</i>. <i>fumigatus fkbp12-3</i> (485 bp) was replaced by the 3.0 kb <i>A</i>. <i>parasiticus pyrG</i> gene. Four of the strains validated by PCR were then selected for Southern analyses. SacI-digested genomic DNA was probed with the 446 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~3.9 kb length as opposed to the wild-type length of ~1.4 kb. Gel used for Southern analysis was 1% agarose. <b>(D) Construction of Δ<i>fkbp12-4</i> strain.</b> In the Δ<i>fkbp12-4</i> mutant, wild-type <i>A</i>. <i>fumigatus fkbp12-4</i> (1653 bp) was replaced by the 3.0 kb <i>A</i>. <i>parasiticus pyrG</i> gene. Four of the strains validated by PCR were then selected for Southern analyses. BamHI-digested genomic DNA was probed with the 677 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~4.5 kb length as opposed to the wild-type length of ~2.0 kb. Gel used for Southern analysis was 1% agarose. <b>(E) Construction of Δ<i>fkbp12-1</i>Δ<i>fkbp12-2</i> strain</b>. In the Δ<i>fkbp12-1</i>Δ<i>fkbp12-2</i> strain, wild-type <i>A</i>. <i>fumigatus fkbp12-2</i> (709 bp) is replaced by the 4.4 kb hygromycin B resistance cassette in the Δ<i>fkbp12-1</i> strain. Four of the strains validated by PCR were then selected for Southern analyses. BamHI-digested genomic DNA was probed with the 550 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~5.2 kb as opposed to the wild-type length of ~1.9 kb.</p

Δ<i>fkbp12-1</i> is resistant to FK506 but its response to other antifungal agents is unchanged.

<p><b>(A)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM at 37°C for 5 days. <b>(B)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM + 100 ng/mL FK506 at 37°C for 5 days. <b>(C)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM + 10 μg/mL cyclosporine A (CsA) at 37°C for 5 days. <b>(D)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM + 1 μg/mL caspofungin (CSP) at 37°C for 5 days <b>(E)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM + 1 μg/mL caspofungin at 37°C for 5 days <b>(F)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴ conidia) incubated on GMM + 100 ng/mL FK506 + 1 μg/mL caspofungin at 37°C for 5 days.</p

Δ<i>fkbp12-1</i> demonstrates full hyphal growth in response to FK506; Δ<i>fkbp12-4</i> is slightly tolerant to FK506.

<p><b>(A)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴/mL) incubated in RPMI for 24 hours. <b>(B)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴/mL) incubated in RPMI with 100 ng/mL FK506 for 24 hours. <b>(C)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴/mL) incubated in RPMI for 48 hours. <b>(D)</b><i>A</i>. <i>fumigatus</i> conidia (10⁴/mL) incubated in RPMI with 100 ng/mL FK506 for 48 hours.</p

Deletion of FKBP12 encoding genes did not alter virulence of <i>A</i>. <i>fumigatus</i>.

<p>Larvae of the wax moth <i>Galleria mellonella</i> were injected with 5 μl of 1 x 10⁸ spores/ml (a total inoculum of 2 x 10⁵ spores) of the wild type, Δ<i>fkbp12-1</i>, Δ<i>fkbp12-2</i>, Δ<i>fkbp12-3</i>, Δ<i>fkbp12-4</i>, and<i>Δfkbp12-1Δfkbp12-2</i> strains. 20 larvae were included in each arm. Infected larvae were incubated at 37°C with survival scored daily for 5 days.</p

FK506 altered the localization of FKBP12-1 to the hyphal septum.

<p><b>(A)</b> Functionality of the expressed FKBP12-1-EGFP was assessed by comparing the growth of the FKBP12-1-EGFP expression strain with the <i>akuB</i>^<i>KU80</i> strain either in the absence or presence of FK506 (0.1 μg/mL) <b>(B, C)</b> Under normal growth conditions, FKBP12-1 evenly distributes throughout the cytoplasm and is also found in the nucleus at the hyphal tips (panel B) and sub-apical compartment (panel C and panel D) (marked by a white arrow heads in panel B and by red arrowheads in panel C and panel D). It is not seen at the septum (marked by a white arrow in the Fig 7C inset). <b>(C)</b> In the presence of FK506, FKBP12-1 can be seen localized as a double bar on either side of the septa indicating its binding to calcineurin complex at the hyphal septum (marked by a white arrows in the Fig 7D inset).</p